Cysteine protease inhibitors

ABSTRACT

Compounds of the formula I 
     
       
         
         
             
             
         
       
     
     wherein
 
R 1a  is H; and R 1b  is C 1 -C 6 alkyl, Carbocyclyl or Het; or
 
R 1a  and R 1b  together define a saturated cyclic amine with 3-6 ring atoms;
 
R 2a  and R 2b  are independently H, halo, C 1 -C 4 alkyl, C 1 -C 4 haloalkyl or C 1 -C 4 alkoxy, or R 2a  and R 2b  together with the carbon atom to which they are attached form a C 3 -C 6 cycloalkyl;
 
R 3  is a branched C 5 -C 10  alkyl chain, C 2 -C 4 haloalkyl or —CH 2 C 3 -C 7  cycloalkyl;
 
R 4  is C 1 -C 6 alkyl, C 1 -C 6 haloalkyl, C 1 -C 6 alkylamino or C 1 -C 6 dialkylamino;
 
for use in the prophylaxis or treatment of a disorder characterised by inappropriate expression or activation of cathepsin S.

TECHNICAL FIELD

This invention relates to inhibitors of cathepsin S, and their use inmethods of treatment for disorders involving cathepsin S such asautoimmune disorders, allergy and chronic pain conditions.

BACKGROUND TO THE INVENTION

The papain superfamily of cysteine proteases are widely distributed indiverse species including mammals, invertebrates, protozoa, plants andbacteria. A number of mammalian cathepsin enzymes, including cathepsinsB, F, H, K, L, O, S, and W, have been ascribed to this superfamily, andinappropriate regulation of their activity has been implicated in anumber of metabolic disorders including arthritis, muscular dystrophy,inflammation, glomerulonephritis and tumour invasion. Pathogeniccathepsin like enzymes include the bacterial gingipains, the malarialfalcipains I, II, III et seq and cysteine proteases from Pneumocystiscarinii, Trypanosoma cruzei and brucei, Crithidia fusiculata,Schistosoma spp.

In WO 97/40066, the use of inhibitors against Cathepsin S is described.The inhibition of this enzyme is suggested to prevent or treat diseasecaused by protease activity. Cathepsin S is a highly active cysteineprotease belonging to the papain superfamily. Its primary structure is57%, 41% and 45% homologous with human cathepsin L and H and the plantcysteine protease papain respectively, although only 31% homologous withcathepsin B. It is found mainly in B cells, dendritic cells andmacrophages and this limited occurrence suggests the potentialinvolvement of this enzyme in the pathogenesis of degenerative disease.Moreover, it has been found that destruction of Ii by proteolysis isrequired for MHC class II molecules to bind antigenic peptides, and fortransport of the resulting complex to the cell surface. Furthermore, ithas been found that Cathepsin S is essential in B cells for effective Iiproteolysis necessary to render class II molecules competent for bindingpeptides. Therefore, the inhibition of this enzyme may be useful inmodulating class II-restricted immune response (WO 97/40066). Otherdisorders in which cathepsin S is implicated are asthma, chronicobstructive pulmonary disease, endometriosis and chronic pain.

BRIEF DESCRIPTION OF THE INVENTION

According to a first aspect of the invention there is provided acompound of the formula I:

wherein

R^(1a) is H; and

R^(1b) is C₁-C₆alkyl, optionally substituted with 1-3 substituentsindependently selected from:

-   -   halo, hydroxy, cyano, azido, C₁-C₄haloalkyl, C₁-C₄alkoxy,        C₁-C₄haloalkoxy, C₁-C₄alkoxycarbonyl, C₁-C₄alkylcarbonyl, amine,        C₁-C₄alkylamine, C₁-C₄dialkylamine, C₁-C₄alkylsulfonyl,        C₁-C₄alkylsulfonylamino, aminocarbonyl, aminosulphonyl,        Carbocyclyl and Het; or

R^(1b) is Carbocyclyl or Het; or

R^(1a) and R^(1b) together with the N atom to which they are attacheddefine a saturated cyclic amine with 3-6 ring atoms;

-   -   wherein the Carbocyclyl, Het or cyclic amine is optionally        substituted with 1-3 substituents independently selected from        halo, hydroxy, cyano, azido, C₁-C₄alkyl, C₁-C₄haloalkyl,        C₁-C₄alkoxy, C₁-C₄haloalkoxy, C₁-C₄alkoxycarbonyl,        C₁-C₄alkylcarbonyl, amine, C₁-C₄alkylamine, C₁-C₄dialkylamine,        C₁-C₄alkylsulfonyl, C₁-C₄alkylsulfonylamino, aminocarbonyl,        aminosulphonyl, RxOOC—C₀-C₂alkylene (where Rx is H, C₁-C₄alkyl        or C₁-C₄haloalkyl), phenyl, benzyl or        C₃-C₆cycloalkylC₀-C₂alkylene;        -   wherein the phenyl, benzyl or cycloalkyl moiety is            optionally substituted with 1-3 substituents independently            selected from halo, C₁-C₄alkyl, C₁-C₄haloalkyl or            C₁-C₄alkoxy);            R^(2a) and R^(2b) are independently selected from H, halo,            C₁-C₄alkyl, C₁-C₄haloalkyl, C₁-C₄alkoxy, or R^(2a) and            R^(2b) together with the carbon atom to which they are            attached form a C₃-C₆cycloalkyl;            R³ is a C₅-C₁₀ alkyl, optionally substituted with 1-3            substituents independently selected from halo,            C₁-C₄haloalkyl, C₁-C₄alkoxy, C₁-C₄haloalkoxy; or            R³ is a C₂-C₄alkyl chain with at least 2 chloro or 3 fluoro            substituents; or            R³ is C₃-C₇cycloalkylmethyl, optionally substituted with 1-3            substituents independently selected from C₁-C₄alkyl, halo,            C₁-C₄haloalkyl, C₁-C₄alkoxy, C₁-C₄haloalkoxy;            R⁴ is C₁-C₆alkyl, C₁-C₆haloalkyl, C₁-C₆alkylamino,            C₁-C₆dialkylamino;            Het is a stable, monocyclic or bicyclic, saturated,            partially saturated or aromatic ring system containing 1-4            heteroatoms independently selected from O, S and N, each            ring having 5 or 6 ring atoms;            Carbocyclyl is C₃-C₆cycloalkyl, C₅-C₆cycloalkenyl or phenyl;            or a pharmaceutically acceptable salt, hydrate or N-oxide            thereof.

In some embodiments, R^(1a) is H and R^(1b) is C₁-C₄alkyl, such asmethyl, ethyl, isopropyl, t-butyl or preferably methyl, optionallysubstituted with one or more substituents as defined above, preferably1-3 halo (e.g. F) or a C₁-C₄alkoxy (e.g. methoxy) group.

In other embodiments R^(1a) is H and R^(1b) is methyl, cyclopropyl,1-phenylethyl, or a 5 or 6 membered heterocyclic ring containing 1-3nitrogen atoms and 0 or 1 sulphur atoms, the cyclopropyl, phenyl orheterocyclic ring being optionally substituted with up to threesubstituents independently selected from:

-   -   C₁-C₄alkyl, halo, C₁-C₄haloalkyl, C₁-C₄alkoxy, C₁-C₄haloalkoxy,        C₁-C₄alkoxycarbonyl, C₁-C₄alkylcarbonyl, amine, C₁-C₄alkylamine,        C₁-C₄-dialkylamine, C₁-C₄alkylsulfonyl, C₁-C₄alkylsulfonylamino,        aminocarbonyl, aminosulphonyl, RxOOC—C₀-C₂alkylene (where Rx is        H or C₁-C₄alkyl) or C₃-C₆cycloalkylC₀-C₂alkylene or benzyl (the        cycloalkyl, or the phenyl ring of the benzyl being optionally        substituted with 1-3 substituents selected from C₁-C₄alkyl,        halo, C₁-C₄haloalkyl, C₁-C₄alkoxy, C₁-C₄haloalkoxy).

Examples of the 5 or 6 membered aromatic heterocyclyl for R^(1b) includepyridyl or pyrimidyl and especially pyrrolyl, pyrazolyl, imidazolyl,thiazolyl, thiadiazolyl, triazolyl or tetrazolyl, optionally substitutedwith any of which is optionally substituted with C₁-C₄alkyl (e.g. Me),halo (e.g. F), C₁-C₄haloalkyl (e.g. CF₃), C₁-C₄alkoxy (e.g. MeO),C₃-C₆cycloalkylC₀-C₁alkylene (e.g. cyclopropyl or cyclopropylmethyl,benzyl or C₀-C₂alkyleneCOOH and its C₁-C₄alkyl esters. An exemplaryspecies is 1-methyl-pyrazol-5-yl.

Typically according to this embodiment, the heterocyclic ring ispyrrolyl, pyrazolyl, imidazolyl, triazolyl, thiazolyl or thiadiazolyl,any of which is optionally substituted with C₁-C₄alkyl, halo,C₁-C₄haloalkyl, C₁-C₄alkoxy, C₃-C₆cycloalkyl or C₃-C₆cycloalkylmethyl.

Typically according to this embodiment, the heterocyclic ring ispyrazol-1-yl, which is optionally substituted with C₁-C₄alkyl, halo,C₁-C₄haloalkyl or cyclopropyl, preferably C₁-C₄alkyl, such as ethyl orpreferably methyl.

A preferred value for R^(1b) is pyrazol-1-yl which is N-substituted withC₁-C₄alkyl, such as ethyl or methyl.

A further typical value for R^(1b) according to this embodiment, ismethyl or cyclopropyl.

In other embodiments R^(1a) is H and R^(1b) is methyl or ethyl which issubstituted in the 1-position with a cyclic group such as phenyl, orR^(1b) is a monocyclic heterocyclyl such as pyrrolidinyl, piperidinyl,morpholinyl, thiomorpholinyl, piperazinyl, indolinyl, pyranyl,tetrahydropyranyl, tetrahydrothiopyranyl, thiopyranyl, furanyl,tetrahydrofuranyl, thienyl, pyrrolyl, oxazolyl, isoxazolyl, thiazolyl,imidazolyl, pyridinyl, pyrimidinyl, pyrazinyl, pyridazinyl, tetrazolyl,pyrazolyl, indolyl and the like. The phenyl or heterocyclyl isoptionally substituted, for example with 1-3 substituents independentlyselected from hydroxy, amino, C₁-C₄alkyl, halo, C₁-C₄haloalkyl,C₁-C₄alkoxy, amino, C₁-C₄alkylamine, C₁-C₄dialkylamine and the like. Anexemplary species is 1-phenylethyl.

In other embodiments R^(1a) is H and R^(1b) is C₃-C₆cycloalkyl,preferably cyclobutyl or cyclopropyl, optionally substituted as definedabove. Preferably the cycloalkyl is unsubstituted or substituted with1-3 substituents selected from halo (e.g. 1 or 2 fluoro), hydroxy,C₁-C₄alkyl (e.g. 1 or 2 methyl), C₁-C₄haloalkyl (e.g. a CF₃ group)C₁-C₄alkoxy (e.g. an MeO group), C₁-C₄alkylamine (e.g. an MeNH— group),C₁-C₄dialkylamine (e.g. an (Me)₂N— group) and the like. An exemplaryspecies is cyclopropyl, or monofluoro- or gemdifluorocyclopropyl.

In some embodiments, R^(1a) is H and R^(1b) is a 6 or preferably 5membered aromatic, heterocyclic ring containing 1-3 nitrogen atoms and 0or 1 sulphur atoms, optionally substituted as defined above. Preferablythe heterocyclic ring is linked to the adjacent nitrogen atom of thealpha keto amide group through a carbon atom of the heterocyclic ring.Exemplary substituents include C₁-C₄alkyl, halo, C₁-C₄haloalkyl,C₁-C₄alkoxy, C₁-C₄haloalkoxy, C₁-C₄alkoxycarbonyl, C₁-C₄alkylcarbonyl,amine, C₁-C₄alkylamine, diC₁-C₄alkylamine, C₁-C₄alkylsulfonyl,C₁-C₄alkylsulfonylamino, aminocarbonyl, aminosulphonyl,RxOC(═O)C₀-C₂alkylene (where Rx is H or C₁-C₄alkyl) orC₃-C₆cycloalkylC₀-C₂alkylene or benzyl (the cycloalkyl or the phenylring of benzyl group) being optionally substituted with 1-3 substituentsselected from C₁-C₄alkyl, halo, C₁-C₄haloalkyl, C₁-C₄alkoxy,C₁-C₄haloalkoxy)

In some embodiments, R^(1a), R^(1b) and the N-atom to which they areattached form a 3-6 membered cyclic amine, such as aziridine, azetidine,pyrrolidine, and preferably morpholine, piperazine or piperidine. Thesecyclic amines may be unsubstituted or substituted as described above,preferably with 1-3 substituents selected from halo (e.g. 1 or 2fluoro), hydroxy, C₁-C₄alkyl (e.g. 1 or 2 methyl), C₁-C₄haloalkyl (e.g.a CF₃ group) C₁-C₄alkoxy (e.g. an MeO— group), C₁-C₄alkylamine (e.g. anMeNH— group), C₁-C₄dialkylamine (e.g. an (Me)₂N— group) and the like.

One embodiment of the invention includes compounds of formula I whereinR^(2a) and R^(2b) are both hydrogen.

In an alternative embodiment, at least one of R^(2a) and R^(2b) is halo,C₁-C₄alkyl, C₁-C₄haloalkyl or C₁-C₄alkoxy. Typically according to theseembodiments, one of R^(2a) and R^(2b) is H, and the other is Cl, F, CF₃or MeO; especially F or MeO.

In a preferred embodiment, one of R^(2a) and R^(2b) is H, and the otheris F. Specially preferred according to this embodiment are compoundshaving the stereochemistry shown in formula (Ia):

In some embodiments, R^(2a) and R^(2b) are both F, thus providingcompounds of formula (Ib):

In other embodiments R^(2a) and R^(2b) together with the carbon atom towhich they are attached form a C₃-C₆cycloalkyl.

In some embodiments R³ is cycloalkylalkyl, optionally substituted, forexample with halo, (such as F) or alkoxy (such as MeO). Exemplaryspecies include 1-methylcyclopentylmethyl, 1-methylcyclohexylmethyl,1-methylcyclobutylmethyl, 1-methyl-3,3-difluorocyclobutylmethyl,1-methyl-4,4-difluorocyclohexylmethyl, cyclopropylmethyl or1-methyl-3,3-difluorocyclopentylmethyl.

Preferred R³ species include t-butylmethyl, cyclobutylmethyl,1-methylcyclobutylmethyl and 1-methylcyclopentylmethyl, any of which isoptionally substituted with one or two F or MeO. Representative speciesare 1-fluorocyclobutylmethyl and 1-fluorocyclopentylmethyl.

Further representative R³ species include 1-methylcyclopentylmethyl and1-fluorocyclopentylmethyl,

Other embodiments have R³ as a straight or branched alkyl chain of 5-10C-atoms, optionally substituted with 1-3 halo, (e.g. Cl or F), or aC₁-C₄alkoxy (e.g. MeO). Exemplary species include 2,2-dimethylpropyl,3,3-dimethylpentyl, 2,2,3,3-tetramethylbutyl. Exemplary species ofhalogenated alkyl include 2,2-dichloroethyl, 3,3,3-trifluoropropyl,2,2-trifluoromethylethyl, or 2,2,2-trifluoroethyl.

One embodiment of the invention include compounds wherein R⁴ isC₁-C₆alkyl, such as methyl or ethyl.

Another embodiment includes compounds wherein R⁴ is C₁-C₆haloalkyl, suchas C₁-C₆chloroalkyl or C₁-C₆fluoroalkyl.

The compounds of formula I are characterised by various advantageouspharmaceutical properties and exhibit at least one improved property inview of the compounds of the prior art. In particular, the inhibitors ofthe present invention are superior in one or more of the followingpharmacological related properties, i.e. potency, decreasedcytotoxicity, improved pharmacokinetics, acceptable dosage and pillburden.

Without in any way wishing to be bound by theory, or the ascription oftentative binding modes for specific variables, P1, P2 and P3 as usedherein are provided for convenience only and have their conventionalmeanings and denote those portions of the inhibitor believed to fill theS1, S2 and S3 subsites respectively of the enzyme, where S1 is adjacentthe cleavage site and S3 remote from the cleavage site.

A further aspect of the invention comprises a method employing thecompounds of formula I for the prophylaxis or treatment of diseasescaused by aberrant expression or activation of cathepsin, i.e. diseasesor conditions alleviated or modified by inhibition of cathepsin S,preferably without substantial concomitant inhibition of other membersof the papain superfamily.

A further aspect of the invention provides the use of the compounds offormula I prophylaxis or treatment of diseases caused by aberrantexpression or activation of cathepsin, ie diseases or conditionsalleviated or modified by inhibition of cathepsin S, preferably withoutsubstantial concomitant inhibition of other members of the papainsuperfamily.

A further aspect of the invention provides the use of the compounds offormula I for the manufacture of a medicament for the prophylaxis ortreatment of diseases caused by aberrant expression or activation ofcathepsin S, i.e. diseases or conditions alleviated or modified byinhibition of cathepsin S, preferably without substantial concomitantinhibition of other members of the papain superfamily.

Examples of such diseases or conditions defined in the immediatelypreceding three paragraphs include those enumerated in WO 97/40066, suchas autoimmune diseases, allergies, such as asthma and hay fever,multiple sclerosis, rheumatoid arthritis and the like. A further exampleis the treatment of endometriasis, and especially chronic pain, asdisclosed in WO03/20287. The invention further provides the use of thecompounds of formula I or any subgroup of formula I in therapy and inthe manufacture of a medicament for the treatment of diseases orconditions alleviated or moderated by inhibition of cathepsin S.

In one series of embodiments, the methods are employed to treat mammals,particularly humans at risk of, or afflicted with, autoimmune disease.By autoimmunity is meant the phenomenon in which the host's immuneresponse is turned against its own constituent parts, resulting inpathology. Many human autoimmune diseases are associated with certainclass II MHC-complexes. This association occurs because the structuresrecognized by T cells, the cells that cause autoimmunity, are complexescomprised of class II MHC molecules and antigenic peptides. Autoimmunedisease can result when T cells react with the host's class II MHCmolecules when complexed with peptides derived from the host's own geneproducts. If these class II MHC/antigenic peptide complexes areinhibited from being formed, the autoimmune response is reduced orsuppressed. Any autoimmune disease in which class II MHC/antigeniccomplexes play a role may be treated according to the methods of thepresent invention.

Such autoimmune diseases include, e.g., juvenile onset diabetes (insulindependent), multiple sclerosis, pemphigus vulgaris, Graves' disease,myasthenia gravis, systemic lupus erythematosus, rheumatoid arthritisand Hashimoto's thyroiditis.

In another series of embodiments, the methods are employed to treatmammals, particularly humans, at risk of, or afflicted with, allergicresponses. By “allergic response” is meant the phenomenon in which thehost's immune response to a particular antigen is unnecessary ordisproportionate, resulting in pathology. Allergies are well known inthe art, and the term “allergic response” is used herein in accordancewith standard usage in the medical field.

Examples of allergies include, but are not limited to, allergies topollen, “ragweed,” shellfish, domestic animals (e.g., cats and dogs),bee venom, house dust mite allergens and the like. Another particularlycontemplated allergic response is that which causes asthma. Allergicresponses may occur, in man, because T cells recognize particular classII MHC/antigenic peptide complexes. If these class II MHC/antigenicpeptide complexes are inhibited from being formed, the allergic responseis reduced or suppressed. Any allergic response in which class IIMHC/antigenic peptide complexes play a role may be treated according tothe methods of the present invention. Immunosuppression by the methodsof the present invention will typically be a prophylactic or therapeutictreatment for severe or life-threatening allergic responses, as mayarise during asthmatic attacks or anaphylactic shock.

In another series of embodiments, the methods are employed to treatmammals, particularly humans, which have undergone, or are about toundergo, an organ transplant or tissue graft. In tissue transplantation(e.g., kidney, lung, liver, heart) or skin grafting, when there is amismatch between the class II MHC genotypes (HLA types) of the donor andrecipient, there may be a severe “allogeneic” immune response againstthe donor tissues which results from the presence of non-self orallogeneic class II MHC molecules presenting antigenic peptides on thesurface of donor cells. To the extent that this response is dependentupon the formation of class II MHC/antigenic peptide complexes,inhibition of cathepsin S may suppress this response and mitigate thetissue rejection. An inhibitor of cathepsin S can be used alone or inconjunction with other therapeutic agents, e.g., as an adjunct tocyclosporin A and/or antilymphocyte gamma globulin, to achieveimmunosuppression and promote graft survival. Preferably, administrationis accomplished by systemic application to the host before and/or aftersurgery. Alternatively or in addition, perfusion of the donor organ ortissue, either prior or subsequent to transplantation or grafting, maybe effective.

The above embodiments have been illustrated with an MHC class IImechanism but the invention is not limited to this mechanism of action.Suppression of cathepsin S as a treatment of COPD or chronic pain maynot, for example, involve MHC class II at all.

A related aspect of the invention is directed to a method of treating apatient undergoing a therapy wherein the therapy causes an immuneresponse, preferably a deleterious immune response, in the patientcomprising administering to the patient a compound of Formula I or apharmaceutically acceptable salt, n-oxide or hydrate thereof. Typically,the immune response is mediated by MHC class II molecules. The compoundof this invention can be administered prior to, simultaneously, or afterthe therapy. Typically, the therapy involves treatment with a biologic,such as a protein, preferably an antibody, more preferably a monoclonalantibody. More preferrably, the biologic is Remicade®, Refacto®,ReferonA®, Factor VIII, Factor VII, Betaseron®, Epogen®, Enbrel®,Interferon beta, Botox®, Fabrazyme®, Elspar®, Cerezyme®, Myobloc®,Aldurazyrne®, Verluma®, Interferon alpha, Humira®, Aranesp®, Zevalin® orOKT3. Alternatively the treatment involves use of heparin, low molecularweight heparin, procainamide or hydralazine.

Assays for the assessment of cathepsin S inhibitors in the treatment ofchronic pain, including neuropathic or inflammatory pain are asdescribed in WO 03/20287.

Currently preferred indications treatable in accordance with the presentinvention include: Psoriasis;

Autoimmune indications, including idiopathic thrombocytopenic purpura(ITP), rheumatoid arthritis (RA), multiple schlerosis (MS), myastheniagravis (MG), Sjögrens syndrome, Grave's disease and systemic lupuserythematosis (SLE);

Non-automimmune indications include allergic rhinitis, asthma,artherosclerosis, chronic obstructive pulmonary disease (COPD) andchronic pain.

The compounds of the invention can form salts which form an additionalaspect of the invention. Appropriate pharmaceutically acceptable saltsof the compounds of the invention include salts of organic acids,especially carboxylic acids, including but not limited to acetate,trifluoroacetate, lactate, gluconate, citrate, tartrate, maleate,malate, pantothenate, isethionate, adipate, alginate, aspartate,benzoate, butyrate, digluconate, cyclopentanate, glucoheptanate,glycerophosphate, oxalate, heptanoate, hexanoate, fumarate, nicotinate,palmoate, pectinate, 3-phenylpropionate, picrate, pivalate, propionate,tartrate, lactobionate, pivolate, camphorate, undecanoate and succinate,organic sulphonic acids such as methanesulphonate, ethanesulphonate,2-hydroxyethane sulphonate, camphorsulphonate, 2-naphthalenesulphonate,benzenesulphonate, p-chlorobenzenesulphonate and p-toluenesulphonate;and inorganic acids such as hydrochloride, hydrobromide, hydroiodide,sulphate, bisulphate, hemisulphate, thiocyanate, persulphate, phosphoricand sulphonic acids.

The compounds of the invention may in some cases be isolated as thehydrate. Hydrates are typically prepared by recrystallisation from anaqueous/organic solvent mixture using organic solvents such as dioxin,tetrahydrofuran or methanol. Hydrates can also be generated in situ byadministration of the corresponding keton to a patient.

The N-oxides of compounds of the invention can be prepared by methodsknown to those of ordinary skill in the art. For example, N-oxides canbe prepared by treating an unoxidized form of the compound of theinvention with an oxidizing agent (e.g., trifluoroperacetic acid,permaleic acid, perbenzoic acid, peracetic acid,meta-chloroperoxybenzoic acid, or the like) in a suitable inert organicsolvent (e.g., a halogenated hydrocarbon such as dichloromethane) atapproximately 0° C. Alternatively, the N-oxides of the compounds of theinvention can be prepared from the N-oxide of an appropriate startingmaterial.

Compounds of the invention in unoxidized form can be prepared fromN-oxides of the corresponding compounds of the invention by treatingwith a reducing agent (e.g., sulphur, sulphur dioxide, triphenylphosphine, lithium borohydride, sodium borohydride, phosphorusdichloride, tribromide, or the like) in an suitable inert organicsolvent (e.g., acetonitrile, ethanol, aqueous dioxane, or the like) at 0to 80° C.

The present invention also includes isotope-labelled compounds offormula I or any subgroup of formula I, wherein one or more of the atomsis replaced by an isotope of that atom, i.e. an atom having the sameatomic number as, but an atomic mass different from, the one(s)typically found in nature. Examples of isotopes that may be incorporatedinto the compounds of formula I or any subgroup of formula I, includebut are not limited to isotopes of hydrogen, such as ²H and ³H (alsodenoted D for deuterium and T for tritium respectively), carbon, such as¹¹C, ¹³C and ¹⁴C, nitrogen, such as ¹³N and ¹⁵N, oxygen, such as ¹⁵O,¹⁷O and ¹⁸O, phosphorus, such as ³¹P and ³²P, sulphur, such as ³⁵S,fluorine, such as ¹⁸F, chlorine, such as ³⁶Cl, bromine such as ⁷⁵Br,⁷⁶Br, ⁷⁷Br and ⁸²Br, and iodine, such as ¹²³I, ¹²⁴I, ¹²⁵I and ¹³¹I.

The choice of isotope included in an isotope-labelled compound willdepend on the specific application of that compound. For example, fordrug or substrate tissue distribution assays, compounds wherein aradioactive isotope such as ³H or ¹⁴C is incorporated will generally bemost useful. For radio-imaging applications, for example positronemission tomography (PET) a positron emitting isotope such as ¹¹C, ¹⁸F,¹³N or ¹⁵O will be useful. The incorporation of a heavier isotope, suchas deuterium, i.e. ²H, may provide greater metabolic stability to acompound of formula I or any subgroup of formula I, which may result in,for example, an increased in vivo half life of the compound or reduceddosage requirements. For example, ²H isotope(s) are typicallyincorporated at position(s) disposed to metabolic liability. In thecompounds of the present invention, suitable positions for incorporationof ²H isotopes are e.g. as substituents to the cyclobutylene group, i.e.one or both of R^(2a) and R^(2b) is ²H.

Isotopically labelled compounds of formula I or any subgroup of formulaI can be prepared by processes analogous to those described in theSchemes and/or Examples herein below by using the appropriateisotopically labelled reagent or starting material instead of thecorresponding non-isotopically labelled reagent or starting material, orby conventional techniques known to those skilled in the art.

It should be noted that the radical positions on any molecular moietyused in the definitions may be anywhere on such moiety as long as it ischemically stable.

As used herein, the following terms have the meanings as defined below:

C_(m)—C_(n)alkyl used on its own or in composite expressions such asC_(m)—C_(n)haloalkyl, C_(m)—C_(n)alkylcarbonyl, C_(m)—C_(n)alkylamine,C_(m)—C_(n)alkylsulphonyl, C_(m)—C_(n)alkylsulfonylamino etc. representsa straight or branched alkyl radical having the number of carbon atomsindicated by m and n, e.g. C₁-C₄alkyl means an alkyl radical having from1 to 4 carbon atoms. Preferred alkyl radicals for use in the presentinvention are C₁-C₄alkyl and includes methyl, ethyl, n-propyl,isopropyl, t-butyl, n-butyl and isobutyl. Methyl and t-butyl aretypically preferred. C₁-C₀alkyl has a corresponding meaning, includingalso all straight and branched chain isomers of pentyl and hexyl. Otherrecitals of C_(m)—C_(n)alkyl, such as C₅-C₁₀ alkyl have thecorresponding meaning.

The term Me means methyl, MeO means methoxy, Et means ethyl and Ac meansacetyl.

C₀-C₂alkylene used in composite expressions such asC₃-C₆cycloalkylC₀-C₂alkylene refers to a divalent radical derived from amethyl or ethyl group, or in the case of C₀ the term C₀-C₂alkylene meansa bond.

C₁-C₄haloalkyl refers to a C₁-C₄alkyl radical, wherein at least one Catom is substituted with a halogen, preferably chloro or fluoro.Trifluoromethyl is typically preferred C₁-C₄alkoxy represents a radicalC₁-C₄alkyl-O wherein C₁-C₄alkyl is as defined above, and includesmethoxy, ethoxy, n-propoxy, isopropoxy, t-butoxy, n-butoxy andisobutoxy. Methoxy and isopropoxy are typically preferred. C₁-C₆alkoxyhas a corresponding meaning, expanded to include all straight andbranched chain isomers of pentoxy and hexoxy. Other recitals ofC_(m)-C_(n)alkoxy, such as C₅-C₁₀alkoxy have the corresponding meaning.

C₁-C₄haloalkoxy as used herein is meant to include C₁-C₄alkoxy whereinat least one C-atom is substituted with one or more halogen atom(s),typically chloro or fluoro. In many cases trifluoromethyl is preferred.

C₁-C₄alkoxycarbonyl means a radical C₁-C₄alkyl-O—C(═O).

Carbocyclyl includes cyclopentyl, cyclohexyl and especially cyclopropyland cyclobutyl. Carbocyclyl further includes cyclopentenyl andcyclohexenyl, in each case with a single double bond. A frequentlypreferred value for Carbocyclyl is phenyl.

Cyclic amine includes aziridine, azetidine, pyrrolidine, piperidine,piperazine and morpholine.

Het is a stable, monocyclic or bicyclic, saturated, partially saturatedor aromatic ring system, containing 1-4 hetero atoms independentlyselected from O, S and N, and each ring having 5 or 6 ring atoms;Exemplary aromatic Het include furan, thiophene, pyrrole, imidazole,pyrazole, triazole, tetrazole, thiazole, oxazole, isoxazole, oxadiazole,thiadiazole, isothiazole, pyridine, pyridazine, pyrazine, pyrimidine,quinoline, isoquinoline, benzofuran, benzothiophene, indole, indazoleand the like. Exemplary unsaturated Het include tetrahydrofuran, pyran,dihydropyran, 1,4-dioxane, 1,3-dioxane, piperidine, pyrrolidine,morpholine, tetrahydrothiopyran, tetrahydrothiophene, 2-H-pyrrole,pyrroline, pyrazoline, imidazoline, thiazolidine, isoxazolidine and thelike.

The compounds of the invention include a number of handles such as OH,NH or COOH groups to which conventional prodrug moieties can be applied.Prodrugs are typically hydrolysed in vivo to release the parent compoundin the plasma, liver or intestinal wall. Favoured prodrugs are esters ofhydroxyl groups such as a phenolic hydroxyl group at R⁴, or aminefunctions such as a sulphonamide amine function. Preferredpharmaceutically acceptable esters include those derived from C₁-C₆carboxylic acids such as acetyl or pivaloyl or optionally substitutedbenzoic acid esters, preferably unsubstituted or substituted withsubstituents broadly as described for R^(1a), typically 1-3 halo (e.g.F), C₁-C₄alkyl (e.g. Me), C₁-C₄haloalkyl (e.g. CF₃) or C₁-C₄alkyloxy(e.g. MeO) groups. Favoured sulphonamide prodrugs include aminoacylsderived from C₁-C₆ carboxylic acids such as acetyl or pivaloyl oroptionally substituted benzoic acid esters, preferably unsubstituted orsubstituted with substituents broadly as described for variable R^(1a),typically 1-3 halo (e.g. F), C₁-C₄alkyl (e.g. Me), C₁-C₄haloalkyl (e.g.CF₃) or C₁-C₄alkyloxy (e.g. MeO) groups.

Unless otherwise mentioned or indicated, the chemical designation of acompound encompasses the mixture of all possible stereochemicallyisomeric forms, which said compound may possess. Said mixture maycontain all diastereomers and/or enantiomers of the basic molecularstructure of said compound. All stereochemically isomeric forms of thecompounds of the present invention both in pure form or mixed with eachother are intended to be embraced within the scope of the presentinvention.

Pure stereoisomeric forms of the compounds and intermediates asmentioned herein are defined as isomers substantially free of otherenantiomeric or diastereomeric forms of the same basic molecularstructure of said compounds or intermediates. In particular, the term“stereoisomerically pure” concerns compounds or intermediates having astereoisomeric excess of at least 80% (i.e. minimum 90% of one isomerand maximum 10% of the other possible isomers) up to a stereoisomericexcess of 100% (i.e. 100% of one isomer and none of the other), more inparticular, compounds or intermediates having a stereoisomeric excess of90% up to 100%, even more in particular having a stereoisomeric excessof 94% up to 100% and most in particular having a stereoisomeric excessof 97% up to 100%. The terms “enantiomerically pure” and“diastereomerically pure” should be understood in a similar way, butthen having regard to the enantiomeric excess, and the diastereomericexcess, respectively, of the mixture in question.

Compounds of the invention can be prepared as their individualstereoisomers by reacting a racemic mixture of the compound with anoptically active resolving agent to form a pair of diastereoisomericcompounds, separating the diastereomers and recovering the opticallypure enantiomer. While resolution of enantiomers can be carried outusing covalent diasteromeric derivatives of compounds of Formula I,dissociable complexes are preferred (e.g., crystalline;diastereoisomeric salts). Diastereomers have distinct physicalproperties (e.g., melting points, boiling points, solubilities,reactivity, etc.) and can be readily separated by taking advantage ofthese dissimilarities. The diastereomers can be separated bychromatography, for example HPLC or, preferably, byseparation/resolution techniques based upon differences in solubility.The optically pure enantiomer is then recovered, along with theresolving agent, by any practical means that would not result inracemization. A more detailed description of the techniques applicableto the resolution of stereoisomers of compounds from their racemicmixture can be found in Jean Jacques Andre Collet, Samuel H. Wilen,Enantiomers, Racemates and Resolutions, John Wiley & Sons, Inc. (1981).

While it is possible for the active agent to be administered alone, itis preferable to present it as part of a pharmaceutical formulation.Such a formulation will comprise the above defined active agent togetherwith one or more acceptable carriers/excipients and optionally othertherapeutic ingredients. The carrier(s) must be acceptable in the senseof being compatible with the other ingredients of the formulation andnot deleterious to the recipient.

The formulations include those suitable for rectal, nasal, topical(including buccal and sublingual), vaginal or parenteral (includingsubcutaneous, intramuscular, intravenous and intradermal)administration, but preferably the formulation is an orally administeredformulation. The formulations may conveniently be presented in unitdosage form, e.g. tablets and sustained release capsules, and may beprepared by any methods well known in the art of pharmacy. Such methodsinclude the step of bringing into association the above defined activeagent with the carrier. In general, the formulations are prepared byuniformly and intimately bringing into association the active agent withliquid carriers or finely divided solid carriers or both, and then ifnecessary shaping the product. The invention extends to methods forpreparing a pharmaceutical composition comprising bringing a compound ofFormula I or its pharmaceutically acceptable salt in conjunction orassociation with a pharmaceutically acceptable carrier or vehicle. Ifthe manufacture of pharmaceutical formulations involves intimate mixingof pharmaceutical excipients and the active ingredient in salt form,then it is often preferred to use excipients which are non-basic innature, i.e. either acidic or neutral.

Formulations for oral administration in the present invention may bepresented as discrete units such as capsules, cachets or tablets eachcontaining a predetermined amount of the active agent; as a powder orgranules; as a solution or a suspension of the active agent in anaqueous liquid or a non-aqueous liquid; or as an oil-in-water liquidemulsion or a water in oil liquid emulsion and as a bolus etc.

With regard to compositions for oral administration (e.g. tablets andcapsules), the term suitable carrier includes vehicles such as commonexcipients e.g. binding agents, for example syrup, acacia, gelatin,sorbitol, tragacanth, polyvinylpyrrolidone (Povidone), methylcellulose,ethylcellulose, sodium carboxymethylcellulose,hydroxypropylmethylcellulose, sucrose and starch; fillers and carriers,for example corn starch, gelatin, lactose, sucrose, microcrystallinecellulose, kaolin, mannitol, dicalcium phosphate, sodium chloride andalginic acid; and lubricants such as magnesium stearate, sodium stearateand other metallic stearates, glycerol stearate stearic acid, siliconefluid, talc waxes, oils and colloidal silica. Flavouring agents such aspeppermint, oil of wintergreen, cherry flavouring or the like can alsobe used. It may be desirable to add a colouring agent to make the dosageform readily identifiable. Tablets may also be coated by methods wellknown in the art.

A tablet may be made by compression or moulding, optionally with one ormore accessory ingredients. Compressed tablets may be prepared bycompressing in a suitable machine the active agent in a free flowingform such as a powder or granules, optionally mixed with a binder,lubricant, inert diluent, preservative, surface-active or dispersingagent. Moulded tablets may be made by moulding in a suitable machine amixture of the powdered compound moistened with an inert liquid diluent.The tablets may be optionally be coated or scored and may be formulatedso as to provide slow or controlled release of the active agent.

Other formulations suitable for oral administration include lozengescomprising the active agent in a flavoured base, usually sucrose andacacia or tragacanth; pastilles comprising the active agent in an inertbase such as gelatine and glycerine, or sucrose and acacia; andmouthwashes comprising the active agent in a suitable liquid carrier.

As with all pharmaceuticals, the appropriate dosage for the compounds orformulations of the invention will depend upon the indication, theseverity of the disease, the size and metabolic vigour and the patient,the mode of administration and is readily determined by conventionalanimal trials. Dosages providing intracellular (for inhibition ofphysiological proteases of the papain superfamily) concentrations of theorder 0.01-100 μM, more preferably 0.01-10 μM, such as 0.1-5 μM aretypically desirable and achievable.

Compounds of the invention are prepared by a variety of solution andsolid phase chemistries.

A typical first step is the preparation of a P1 building block of theformula V

where R^(2a) and R^(2b) are as defined above, PG is a conventional Nprotecting group such as Boc, CBz or Fmoc and PG* is H or a conventionalcarboxy protecting group, such as a C₁-C₄alkyl or benzyl. These buildingblocks are novel and constitute a further aspect of the invention.

Building blocks of formula V are typically prepared as described inscheme 1 below.

A suitable starting material is an N-protected cyclobutyl amino acid, ofwhich several are available commercially or can be prepared as shown inthe following Examples or as described by Allan et al. in J. Med. Chem.,1990 33(10) 2905-2915.

The carboxylic acid (1a) is transformed via a Weinreb synthesis to aN,O-dimethylhydroxamic acid (1b) which provides the correspondingaldehyde (1c). The aldehyde may also be accessed by reduction of thecarboxylic function of a cyclobutyl amino acid and oxidation under DessMartin conditions. The aldehyde (1c) can be subsequently reacted withthe appropriate isocyanide in a Passerini reaction to afford therequired α-hydroxy R^(1a)R^(1b) amide (1d). However, in the case wherethe appropriate isocyanide is not readily available, t-butylisocyanidecan alternatively be used, thus affording the t-butyl amide, which afterhydrolysis of the amide, provides the α-hydroxycarboxylic acid P1building block (1e). Generally the strongly acidic conditions requiredto hydrolyse the amide also lead to loss of the NBoc protection, ifused, hence, the amine can be used directly to couple to a P2 buildingblock or else if it needs to be stored, the amine can be reprotected.

The P1 building block thus afforded is then extended at the C and Ntermini to provide compounds of formula I. In scheme 2, a general routeto compounds wherein R⁴ is C₁-C₆alkyl or C₁-C₆haloalkyl.

Typically the C terminus is extended first by reaction of the buildingblock of formula V (2a) with an R^(1a)*R^(1b)* amine, where R^(1a)* andR^(1b)* are R^(1a) and R^(1b) respectively or synthons therefor(selected in view of the sensitivity of the R^(1b) function for the P3elongation conditions outlined below). The reaction proceeds withconventional peptide chemistries as discussed below. The thus preparedP1-prime side unit (2b) is thereafter deprotected at the N terminus andelongated with the P2 building block, providing the N-protectedintermediate amine 2c, and subsequently P3 building block, providing theamide 2d, using conventional peptide chemistries. For example a P2residue can be introduced via BocP2-OH using standard couplingconditions such as HATU, DIPEA in DMF. The terminal Boc protection isagain removed with acetyl chloride in methanol or equivalent and the P3residue is introduced using standard peptide coupling techniques, suchas by reaction with the appropriate P3-acid using conditions like HATU,DIPEA in DMF or by reaction with a P3-acid halide such as the acidchloride or the like. The final steps will generally comprise conversionof the R^(1a)*/R^(1b)* synthons (if present) to their final form andfinally oxidation of the alpha hydroxy amide function using Dess Martinconditions to provide the desired alpha keto amide compound 2e.

An extensive range of appropriately protected L-amino acids suitable forP2 building blocks and carboxylic acids, carboxylic acid halides andcarbamoyl halides suitable for P3 building blocks are commerciallyavailable or accessed by simple chemistries or as shown in WO06/064286.The P3 and P2 building blocks may alternatively be coupled first andthen reacted with the P1-prime side unit.

Elongation is typically carried out in the presence of a suitablecoupling agent e.g., benzotriazole-1-yloxytrispyrrolidinophosphoniumhexafluorophosphate (PyBOP),O-benzotriazol-1-yl-N,N,N′,N′-tetramethyl-uronium hexafluorophosphate(HBTU), O-(7-azabenzotriazol-1-yl)-1,1,3,3-tetramethyl-uroniumhexafluorophosphate (HATU),1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC), or1,3-dicyclohexyl carbodiimide (DCC), optionally in the presence of1-hydroxybenzotriazole (HOBT), and a base such asN,N-diisopropylethylamine, triethylamine, N-methylmorpholine, and thelike. The reaction is typically carried out at 20 to 30° C., preferablyat about 25° C., and requires 2 to 24 h to complete. Suitable reactionsolvents are inert organic solvents such as halogenated organic solvents(e.g., methylene chloride, chloroform, and the like), acetonitrile,N,N-dimethylformamide, ethereal solvents such as tetrahydrofuran,dioxane, and the like.

Alternatively, the above elongation coupling step can be carried out byfirst converting the P3/P2 building block into an active acid derivativesuch as succinimide ester and then reacting it with the P1 amine. Thereaction typically requires 2 to 3 h to complete. The conditionsutilized in this reaction depend on the nature of the active acidderivative. For example, if it is an acid chloride derivative, thereaction is carried out in the presence of a suitable base (e.g.triethylamine, diisopropylethylamine, pyridine, and the like). Suitablereaction solvents are polar organic solvents such as acetonitrile,N,N-dimethylformamide, dichloromethane, or any suitable mixturesthereof.

The term “N-protecting group” or “N-protected” as used herein refers tothose groups intended to protect the N-terminus of an amino acid orpeptide or to protect an amino group against undesirable reactionsduring synthetic procedures. Commonly used N-protecting groups aredisclosed in Greene, “Protective Groups in Organic Synthesis” (JohnWiley & Sons, New York, 1981), which is hereby incorporated byreference. N-protecting groups include acyl groups such as formyl,acetyl, propionyl, pivaloyl, t-butylacetyl, 2-chloroacetyl,2-bromoacetyl, trifluoracetyl, trichloroacetyl, phthalyl,o-nitrophenoxyacetyl, α-chlorobutyryl, benzoyl, 4-chlorobenzoyl,4-bromobenzoyl, 4-nitrobenzoyl, and the like; sulfonyl groups such asbenzenesulfonyl, p-toluenesulfonyl, and the like, carbamate forminggroups such as benzyloxycarbonyl, p-chlorobenzyloxycarbonyl,p-methoxybenzyloxycarbonyl, p-nitrobenzyloxycarbonyl,2-nitrobenzyloxycarbonyl, p-bromobenzyloxycarbonyl,3,4-dimethoxybenzyloxycarbonyl, 4-methoxybenzyloxycarbonyl,2-nitro-4,5-dimethoxybenzyloxycarbonyl,3,4,5-trimethoxybenzyloxycarbonyl,1-(p-biphenylyl)-1-methylethoxycarbonyl,α,α-dimethyl-3,5-dimethoxybenzyloxycarbonyl, benzhydryloxycarbonyl,t-butoxycarbonyl, diisopropylmethoxycarbonyl, isopropyloxycarbonyl,ethoxycarbonyl, methoxycarbonyl, allyloxycarbonyl,2,2,2-trichloroethoxycarbonyl, phenoxycarbonyl, 4-nitrophenoxycarbonyl,fluorenyl-9-methoxycarbonyl, cyclopentyloxycarbonyl,adamantyloxycarbonyl, cyclohexyloxycarbonyl, phenylthiocarbonyl, and thelike; alkyl groups such as benzyl, triphenylmethyl, benzyloxymethyl andthe like; and silyl groups such as trimethylsilyl and the like. FavouredN-protecting groups include formyl, acetyl, benzoyl, pivaloyl,t-butylacetyl, phenylsulfonyl, benzyl (bz), t-butoxycarbonyl (BOC) andbenzyloxycarbonyl (Cbz).

Hydroxy and/or carboxy protecting groups are also extensively reviewedin Greene ibid and include ethers such as methyl, substituted methylethers such as methoxymethyl, methylthiomethyl, benzyloxymethyl,t-butoxymethyl, 2-methoxyethoxymethyl and the like, silyl ethers such astrimethylsilyl (TMS), t-butyldimethylsilyl (TBDMS) tribenzylsilyl,triphenylsilyl, t-butyldiphenylsilyl triisopropyl silyl and the like,substituted ethyl ethers such as 1-ethoxymethyl,1-methyl-1-methoxyethyl, t-butyl, allyl, benzyl, p-methoxybenzyl,dipehenylmethyl, triphenylmethyl and the like, aralkyl groups such astrityl, and pixyl (9-hydroxy-9-phenylxanthene derivatives, especiallythe chloride). Ester hydroxy protecting groups include esters such asformate, benzylformate, chloroacetate, methoxyacetate, phenoxyacetate,pivaloate, adamantoate, mesitoate, benzoate and the like. Carbonatehydroxy protecting groups include methyl vinyl, allyl, cinnamyl, benzyland the like.

DETAILED DESCRIPTION OF THE EMBODIMENTS

Various embodiments of the invention will now be described by way ofillustration only with reference to the following Examples.

In the examples below, the following systems are typically employed:

Nuclear Magnetic Resonance (NMR) spectra were recorded on a VarianGemini 7 Tesla 300 MHz instrument, or a Bruker Avance 400 MHz instrumentin the solvent indicated. Chemical shifts are given in ppm down- andupfield from tetramethylsilane (TMS). Resonance multiplicities aredenoted s, d, t, m, br and app for singlet, doublet, triplet, multiplet,broad and apparent, respectively. The Mass Spectrometry (MS) spectrawere recorded on a Finnigan SSQ7000 TSP or a Finnigan SSQ710 DI/EIinstrument. LC-MS was obtained with a Waters 2790 LC-system equippedwith a Waters Xterra™ MS C₈ 2.5 μam 2.1×30 mm column, a Waters 996Photodiode Array Detector and a Micromass ZMD. High pressure liquidchromatography (HPLC) assays were performed using a Hewlett Packard 1100Series HPLC system equipped with a Zorbax column SB-C₈ 4.6 mm×15 cm.Column chromatography was performed using silica gel 60 (230-400 meshASTM, Merck) and thin layer chromatography (TLC) was performed on TLCprecoated plates, silica gel 60 F₂₅₄ (Merck).

Preparation of Building Block 1, a P1 Building Block

Step a) [1-(Methoxy-methyl-carbamoyl)-cyclobutyl]-carbamic acidtert-butyl ester (BB1-a)

To a solution of 1-tert-butoxycarbonylamino-cyclobutanecarboxylic acid(3 g, 13.94 mmol) in dry DMF (50 mL) was added N,O-dimethylhydroxylaminex HCl (1.36 g, 13.94 mmol) and DIEA (9.21 mL, 55.75 mmol). The reactionflask was cooled to 0° C. and after 10 minutes HATU (5.30 g, 13.94 mmol)was added to the solution (which turned yellow on addition). After 2 hrsthe DMF was removed by rotary evaporation at reduced pressure. Theresidue was dissolved in EtOAc (100 mL) and washed twice with 10% citricacid (aq) and saturated NaHCO₃(aq) solution. The organic phase was driedwith Na₂SO₄, filtered and evaporated on silica. The product was purifiedby flash chromatography (heptane: ethyl acetate (1:1) to give theproduct as a colourless oil that slowly crystallizes (3.13 g) in 87%yield.

Step b) (1-Formyl-cyclobutyl)-carbamic acid tert-butyl ester (BB1-b)

LiAlH₄ (202 mg, 5.33 mmol) was added to a solution of the Weinreb amideBB1-a (1.10 g, 4.27 mmol) dissolved in dry diethyl ether (35 mL) at 0°C. The solution was stirred at 15 minutes before the reaction wasquenched with slow addition of potassium hydrogen tartaric acid (sat,aq) and stirred for 10 minutes. The solution was poured into aseparatory funnel and the water phase was extracted with ethyl acetatetwice. The combined organic phases were washed with 0.5 M HCl (3 times),NaHCO₃(aq) (2 times) and brine (1 time). The organic phase was driedwith Na₂SO₄, filtered and evaporated on silica. The product was purifiedby flash chromatography (heptane: ethyl acetate (4:1→3:1) to give theproduct as white crystals (0.647 g) in 76% yield.

Step c) [1-(tert-Butylcarbamoyl-hydroxy-methyl)-cyclobutyl]-carbamicacid tert-butyl ester (BB1-c)

BB1-b, (1.75 g, 8.78 mmol) was dissolved in CH₂Cl₂ (18 mL) and cooled inan ice bath, under inert gas. Pyridine (2.85 mL) was added, followed byt-butyl isocyanide (1.50 mL, 13.3 mmol). Trifluoroacetic acid (1.35 mL,17.5 mmol) was then added dropwise over 30 min. The yellow solution wasstirred at RT overnight. The mixture was concentrated, diluted withEtOAc (100 mL) and washed successively with 1N HCl (50 mL), saturatedNaHCO₃ (50 mL) and saturated NaCl (2×50 mL). Drying (Na₂SO₄) andconcentration under vacuum. The afforded crude product was treated withTHF (2.5 mL) and 1M LiOH in 3/1 MeOH-water (2.5 mL) at RT. TLC (3/1petroleum ether—EtOAc) showed complete ester hydrolysis after 15 min.After 45 min reaction time, 1N HCl (2.5 mL), water (10 mL) and EtOAc (20mL) were added, and the layers were separated. The organic phase waswashed with saturated NaHCO₃ (20 mL) and then saturated NaCl (2×20 mL),dried (Na₂SO₄) and concentrated. Flash chromatography (75 g silica, 5/1to 1/1 petroleum ether—EtOAc) gave a white solid (2.36 g, 89%).

Step d (1-Tert-butoxycarbonylamino-cyclobutyl)-hydroxyacetic acid (BB1)

BB1-c (1.30 g, 4.33 mmol) was refluxed with 6N HCl (40 mL) until amidehydrolysis was complete as monitored by LCMS. The mixture wasevaporated, co-evaporating several times with water. 1M NaOH (15 mL) wasadded to the residue and the basic solution was stirred under vacuum for15 min. Boc₂O (1.92 g, 8.80 mmol) in dioxane (10 mL) was added, keepingpH at 10-11, and the mixture was stirred at RT overnight. The mixturewas diluted with water (50 mL), acidified with 1N HCl to pH 3, in an icebath, and then extracted with EtOAc (2×50 mL, then 30 mL). The organicphase was washed with saturated NaCl (50 mL), dried (Na₂SO₄) andevaporated to give crude P1 building block BB1 (0.649 g).

^(1H)NMR (400 MHz, d₆-DMSO) δ 6.88 (br s, 1H), 4.15 (s, 1H), 2.40 (br m,2H), 1.98 (br m, 2H), 1.80 (br m, 2H), 1.35 (s, 9H); ms ES⁺ m/z 146(100%), 190 (50%).

Preparation of Building Block 2, a P1 Building Block

Step a) ((1-Bromo-3-chloropropan-2-yloxy)methyl)benzene (BB2-a)

To a stirred mixture of benzyl bromide (185 g, 1.08 mol) and (1.5 g) ofmercurous chloride was added epichlorohydrin (100 g, 1.08 mol). Thereaction mixture was heated for 12 hr at 100° C. TLC analysis confirmedformation of product. The product was separated from the dark brownreaction mixture by column chromatography using petroleum ether aseluent. TLC system; Petroleum ether: ethyl acetate (9:1), R_(F)=0.7.Yield; 148 g, 51%.

Step b) 3-Benzyloxy-cyclobutane-1,1-dicarboxylic acid diethyl ester(BB2-b)

To a stirred suspension of sodium hydride (22.5 g, 0.562 mol) in 800 mLof dry dioxane, was added diethyl malonate (90 g, 0.562 mol) drop-wiseover 20 min. After this addition was complete, BB2-a (148 g, 0.56 mol)was added drop-wise over 20 min. The mixture was then heated at refluxfor 24 hr. After cooling to room temperature, sodium hydride (22.5 g,0.562 mol) in a little dioxane (˜20 mL) was added to the mixture andheating at reflux was continued for an additional 48 hr. The solvent waspartially removed under reduced pressure and the mixture was treatedwith 800 mL of water. This mixture was then extracted with ethyl acetate(500 mL×3), extracts were dried (Na₂SO₄) and concentrated in vacuo andthe residue was purified by column chromatography using petroleum ether:ethyl acetate (10%) which gave the title compound. TLC system; petroleumether: ethyl acetate (9:1), R_(F)=0.3. Yield: 100 g, 58%

Step c) Diethyl 3-hydroxycyclobutane-1,1-dicarboxylate (BB2-c)

To a solution of compound BB2-b (40 g) in EtOH (500 mL) was added 10%palladium on charcoal (4 g) and the mixture was hydrogenated for 3.5hours at 50 psi at room temperature. The catalyst was removed byfiltration, washed with ethyl acetate, EtOH and the solvent was thenremoved under reduced pressure. The residue was purified by silica gelchromatography with hexane/ethyl acetate as eluent to provide the titlecompound. TLC system; Petroleum ether: ethyl acetate (9:1), R_(F)=0.3.Yield: 18 g, 64%.

Step d) Diethyl 3-oxocyclobutane-1,1-dicarboxylate (BB2-d)

To a solution of compound BB2-c (18 g, 0.0833 mol) in DCM (200 mL) wasadded PCC (37 g, 0.176 mol) and the mixture was stirred for four hoursat room temperature. The solution was filtered through a silica gelcolumn and the residue was washed with DCM/MeOH 98/2 and then filteredthrough a similar column. The combined fractions were evaporated underreduced pressure to provide the desired compound. TLC system; Petroleumether: ethyl acetate (9:1), R_(F)=0.3. Yield: 11 g, 62%.

Step e) Diethyl 3,3-difluorocyclobutane-1,1-dicarboxylate (BB2-e)

To a cooled solution of compound BB2-d (11 g, 0.0513 mol) in dry DCM(150 mL) was added drop-wise a solution of DAST (18.72 g, 0.116 mol) andthe mixture was stirred at room temperature overnight. The mixture wasadded to ice water and was extracted three times with DCM. The solutionwas dried with sodium sulphate and evaporated under reduced pressure.The residue was purified by silica gel chromatography employinghexane/ethyl acetate as eluent to provide the title compound. TLCsystem; Petroleum ether: ethyl acetate (7:3), R_(F)=0.5. Yield: 7.7 g,64%.

Step f) 1-(Ethoxycarbonyl)-3,3-difluorocyclobutanecarboxylic acid(BB2-f)

Compound BB2-e (7.7 g, 0.0325 mol) was dissolved in ice cooled 0.5 Methanolic potassium hydroxide solution (30 mL) and water (6 mL). Themixture was stirred at room temperature overnight. Water was added andmost of the ethanol was removed under reduced pressure. The mixture wasacidified with 2M HCl and extracted three times with ethyl acetate. Theorganic phase was dried with sodium sulphate and evaporated underreduced pressure to give the desired compound. TLC system: petroleumether: ethyl acetate (1:1), R_(F)=0.3. Yield: 5.8 g, 86%.

Step g) Ethyl1-(tert-butoxycarbonylamino)-3,3-difluorocyclobutanecarboxylate (BB2-g)

To a solution of compound BB2-f (5.8 g, 0.0273 mol) in dry dioxane (100mL) was added tert-butanol (24.4 mL), DPPA (7.87 g, 0.027 mol) and TEA(2.87 g, 0.0284 mol) and the mixture was refluxed for five hours. Ethylacetate (about 200 mL) was added and the organic phase was washed twicewith 5% citric acid and saturated sodium hydrogen carbonate. Thesolution was dried and evaporated under reduced pressure. The desiredproduct was isolated by silica gel chromatography with hexane/ethylacetate. TLC system; Petroleum ether: ethyl acetate (1:1), R_(F)=0.5.Yield: 4 g, 51.4%.

Step h) tert-Butyl 3,3-difluoro-1-(hydroxymethyl)cyclobutylcarbamate(BB2-h)

To a ice cooled solution of compound BB2-g (4 g, 0.0143 mol) in dry THF(100 mL) was slowly added a solution of 2M lithium borohydride (30 mL)and the mixture was allowed to warm up to room temperature. The mixturewas stirred for three hours at room temperature. Ice water and 5% citricacid were added and the mixture was extracted three times with DCM. Theorganic phase was dried (Na₂SO₄), filtered and evaporated under reducedpressure which gave the title compound. TLC system petroleum ether:ethyl acetate (1:1), R_(F)=0.3. Yield: 3.1 g, 91%.

Step i) tert-Butyl 3,3-difluoro-1-formylcyclobutylcarbamate (BB2-i)

To a solution of compound BB2-h (3.1 g, 0.0130 mol) in dry DCM (100 mL)was added Dess Martin Period inane (19.9 g, 0.0470 mol) and the mixturewas stirred for three hours at room temperature. Ethyl acetate (200 mL)was added and the organic phase was washed twice with 10% sodiumthiosulphate solution, twice with 0.5 M NaOH and with brine. The organicphase was dried and evaporated under reduced pressure. The residue waspurified by silica gel chromatography with hexane/ethyl acetate aseluent which gave the title compound. TLC system; petroleum ether: ethylacetate (1:1), R_(F)=0.4. Yield: 2.7 g, 87%.

Step j) tert-Butyl1-(2-(tert-butylamino)-1-hydroxy-2-oxoethyl)-3,3-difluorocyclobutylcarbamate(BB2-j)

To a ice cooled solution of compound BB2-i (1.5 g, 0.0064 mol) in dryDCM (100 mL) was added tert-butylisocyanate (0.81 g, 0.009 mol) andpyridine (2.04 g, 0.027 mol). Trifluoroacetic acid (1.58 g, 0.015 mol)was added over a ten minutes period. The mixture was stirred for fivehours at room temperature. Ethyl acetate was added and the organic phasewas washed twice with 5% citric acid and brine. The organic phase wasevaporated and dissolved in dioxane (50 mL). 1M LiOH solution (100 mL)was added and the mixture was stirred overnight at room temperature. 5%Citric acid was added and the mixture was extracted three times withethyl acetate. The organic phase was washed with brine, dried (Na₂SO₄),filtered and evaporated under reduced pressure. The product was purifiedby silica gel chromatography with hexane/ethyl acetate as eluent. TLCsystem; Petroleum ether: ethyl acetate (1:1), R_(F)=0.4. Yield: 1.0 g,46%.

Step k)2-(1-(Tert-Butoxycarbonylamino)-3,3-difluorocyclobutyl)-2-hydroxyaceticacid (BB2)

Compound BB2-j (1 g) was dissolved in 6N HCl (40 mL), and heated toreflux for 24 h after which TLC showed that the reaction had reachedcompletion. The reaction mixture was concentrated in vacuo and residuewas dissolved in THF; H₂O (7; 3, 50 mL), and TEA (1.8 mL, 0.012 mol) andBoc anhydride (2.6 g, 0.012 mol) were both added. The mixture wasstirred at RT for 8 h when TLC confirmed the reaction had reachedcompletion. The reaction mixture was concentrated in vacuo and theresidue was purified by column chromatography using 5% methanol inchloroform which gave the title compound. TLC system; MeOH: CHCl₃ (1:9),R_(F)=0.4. Yield: 0.6 g, 72%.

^(1H) NMR (400 MHz, d6-DMSO) δ 7.30 (br s, 1H), 4.11 (s, 1H), 2.90 (brm, 2H), 2.61 (br m, 2H), 1.35 (s, 9H); ms ES⁺ m/z 281 (100%).

Preparation of Building Block 3—a P1 Building Block

Step a) ((1-Bromo-3-chloropropan-2-yloxy)methyl)benzene (BB3-a)

A mixture of benzyl bromide (46.0 g, 0.269 mol) and epichlorohydrin(24.9 g, 0.269 mol) and mercurous chloride (0.04 g, 0.085 mmol) washeated for 12 h at 150° C. The crude product was purified by columnchromatography (silica gel 60-120 mesh, eluent 1% EtOAc in pet ether)which afforded the title compound as a viscous liquid (50 g, yield 70%).TLC system: 10% EtOAc in pet ether, R_(f)=0.6.

Step b) Diethyl 3-(benzyloxy)cyclobutane-1,1-dicarboxylate (BB3-b)

In a three-neck flask equipped with stirrer, additional funnel andreflux condenser was place NaH (4.6 g, 0.192 mol) in dry dioxane (150mL). To this stirred reaction mixture, diethyl malonate (30.75 g, 0.192mol) was added drop-wise over 30 min. After the addition was complete,compound BB3-a (50 g, 0.19 mol) was added drop-wise over a period of 30min. The reaction mixture was refluxed for 24 h. After cooling to roomtemperature, NaH (4.6 g, 0.192 mol) and dry dioxane (40 mL) was added tothe reaction mixture and further heated to reflux for another 48 h. Thesolvent was partially removed under reduced pressure and the mixture wastreated with water (150 mL). The product was extracted with diethylether (3×100 mL), the organic layer was washed with brine and dried overanhydrous Na₂SO₄. Solvent was concentrated in vacuum and the crudeproduct was purified by column chromatography (silica gel 60-120 mesh,eluent 2% EtOAc in pet ether) which afforded the title compound as aviscous liquid (33 g, yield 57%). TLC system: 15% EtOAc in pet ether,R_(f)=0.5.

Step c) Diethyl 3-hydroxycyclobutane-1,1-dicarboxylate (BB3-c)

To a solution of compound BB3-b (33 g, 0.108 mol) in EtOH (300 mL) wasadded 10% palladium on charcoal (10 g) and the mixture was hydrogenatedfor 48 h with 50 psi pressure at room temperature. The catalyst wasremoved by filtration through a celite bed and washed thoroughly withEtOAc. The solvent was removed under reduced pressure. The product waspurified by silica gel chromatography (silica gel 60-120 mesh, eluent20% EtOAc in pet ether) which afforded the title compound as a viscousliquid (12 g, yield 51%). TLC system: 30% EtOAc in pet ether, R_(f)=0.3.

Step d) Diethyl 3-fluorocyclobutane-1,1-dicarboxylate (BB3-d)

Compound BB3-c (0.8 g, 0.0037 mol) was dissolved in dry DCM (16 mL) andcooled to 0° C. DAST (1.8 g, 0.011 mol) was added drop-wise to the coldsolution. The reaction mixture was warmed to room temperature stirredfor 12 h. The reaction mixture was quenched with cold saturated NaHCO₃solution. The crude product was extracted with DCM (100 mL). The organiclayer was washed with 10% NaHCO₃ solution, water followed by brine anddried over anhydrous Na₂SO₄. Solvent was concentrated in vacuum and thecrude product was purified by column chromatography (silica gel 60-120mesh, eluent 1-2% EtOAc in pet ether) which afforded the title compoundas a pale yellow liquid (460 mg, yield 57%). TLC system: 10% EtOAc inpet ether, R_(f)=0.4.

Step e) 1-(Ethoxycarbonyl)-3-fluorocyclobutanecarboxylic acid (BB3-e)

Compound BB3-d (0.46 g, 0.0021 mol) was dissolved in ice cooled 0.5Mpotassium hydroxide solution in EtOH (4.2 mL) and water (1.4 mL). Themixture was stirred at room temperature overnight. Water was added andmost of the ethanol was removed under reduced pressure. The mixture wasacidified with 2N HCl and extracted with EtOAc (3×50 mL). The organicphase was dried over anhydrous Na₂SO₄. Solvent was concentrated invacuum to afford the crude title compound (0.35 g, crude) which was usedas such for the next step. TLC system: 50% EtOAc in pet ether,R^(f)=0.3.

Step f) Ethyl1-(tert-butoxycarbonylamino)-3-fluorocyclobutanecarboxylate (BB3-f)

To a solution of compound BB3-e (0.35 g, 0.0018 mol) in dry dioxane (6mL) was added tert-butanol (1.8 mL), diphenyl phosphoryl azide (0.56 g,0.002 mol) and triethylamine (0.2 g, 0.002 mol) and the mixture wasrefluxed for 5 h. After completion of the reaction, EtOAc (60 mL) wasadded to the reaction mixture and the organic layer was washed with 5%citric acid (2×20 mL) followed by saturated NaHCO₃ (50 mL). The organicsolvent was evaporated under reduced pressure. To the residue EtOAc (100mL) was added and the organic layer was washed with brine and dried overanhydrous Na₂SO₄. Solvent was concentrated in vacuum and the crudeproduct was purified by column chromatography (silica gel 60-120 mesh,eluent 5-10% EtOAc in pet ether) which afforded the title compound aswhite crystals (0.27 g, yield 56%). TLC system: 20% EtOAc in pet ether,R_(f)=0.4.

Step g) tert-Butyl 3-fluoro-1-(hydroxymethyl)cyclobutylcarbamate (BB3-g)

To a ice cooled solution of compound BB3-f (0.27 g, 0.001 mol) in dryTHF (10 mL) was slowly added a solution of 2M lithium borohydride (2 mL,0.004 mol) and the mixture was allowed to warm up to room temperature.The mixture was stirred for 3 h at room temperature. The reactionmixture was quenched with ice water (2 mL) and 5% citric acid (5 mL) andthe crude product was extracted with DCM (2×50 mL). The organic layerwas washed with brine and dried over anhydrous Na₂SO₄. Solvent wasconcentrated in vacuum and the crude product was purified by columnchromatography (silica gel 60-120 mesh, eluent 15-18% EtOAc in petether) which afforded the title compound as white solid (90 mg, yield39%). TLC system: 50% EtOAc in pet ether, R_(f)=0.5.

Step h) tert-Butyl 3-fluoro-1-formylcyclobutylcarbamate (BB3-h)

To a degassed solution of compound BB3-g (90 mg, 0.0004 mol) in dry DCM(4.5 mL) was added Dess-Martin Periodinane (0.21 g, 0.0005 mol) and themixture was stirred for 3 h at room temperature. EtOAc (30 mL) was addedand the organic layer was washed with 10% sodium thiosulphate solution(2×10 mL), 0.5 M NaOH (20 mL) and with brine. The organic layer wasdried over anhydrous Na₂SO₄. Solvent was concentrated in vacuum and thecrude product was purified by column chromatography (silica gel 60-120mesh, eluent 10-15% EtOAc in pet ether) which afforded the titlecompound as a white crystalline solid (75 mg, yield 87%). TLC system:20% EtOAc in pet ether, R_(f)=0.4.

Step i) tert-Butyl1-(2-(tert-butylamino)-1-hydroxy-2-oxoethyl)-3-fluorocyclobutylcarbamate(BB3-i)

To an ice cooled solution of compound BB3-h (1.3 g, 0.0059 mol) in dryDCM (25 mL) was added tert-butyl isocyanide (0.75 g, 0.0089 mol) and drypyridine (2.6 mL). Trifluoroacetic acid (0.9 mL, 0.0118 mol) was addedover a period of ten minutes maintaining the temperature at 0° C. Thereaction mixture was slowly warmed to room temperature and stirred for16 h. EtOAc (50 mL) was added and the organic phase was washed twicewith 5% citric acid and brine. The organic phase was evaporated and thecrude product was dissolved in THF (25 mL). 1M LiOH solution in MeOH—H₂O(3:2v/v) (2.6 mL) was added and the mixture was stirred for 2 h at roomtemperature. The reaction mixture was quenched with 5% citric acid andthe mixture was extracted with ethyl acetate (2×25 mL). The organiclayer was washed with brine and dried over anhydrous Na₂SO₄. Solvent wasevaporated in vacuum and to afford the title compound which was pureenough to be used in the next step (1.6 g, yield 84%). TLC system: 20%EtOAc in pet ether, R_(f)=0.3.

Step j)2-(1-(tert-Butoxycarbonylamino)-3-fluorocyclobutyl)-2-hydroxyacetic acid(BB3)

Compound BB3-i (1.6 g, 0.005 mol) was refluxed with 6N HCl (60 mL) for16 h until the amide hydrolysis was complete. The solvent was evaporatedunder reduced pressure and co-evaporated several times with water. Theproduct was dissolved in THF:H₂O (7:3 v/v, 50 mL), cooled to 0° C. andEt₃N (2.1 mL, 0.015 mol) was added followed by di-tert-butyl dicarbonate(2.18 g, 0.01 mol). The mixture was stirred at room temperatureovernight (pH was monitored in a regular interval and kept ˜11throughout the reaction). The reaction mixture was neutralized with 1NHCl and the product was extracted with EtOAc (2×50 mL). The organiclayer was washed with brine and dried over anhydrous Na₂SO₄. The solventwas evaporated under reduced pressure followed by purification by columnchromatography (silica gel 60-120 mesh, eluent 5% MeOH in CHCl₃) whichafforded the title P1 building block as a solid (0.65 g, yield 50%). TLCsystem: 30% MeOH in CHCl₃, R_(f)=0.3.

¹H NMR (400 MHz, d₆-DMSO) δ 7.01 (br s, 1H), 5.16 (br m, 1H), 4.97 (brm, 1H), 2.49 (br m, 5H), 1.36 (s, 9H); ms ES⁺ m/z 262 (100%).

Building Block 4—a P1-Prime Side Building Block

Step a) tert-butyl 1-(hydroxymethyl)-3-methoxycyclobutylcarbamate(BB4-a)

500 mg (1.51 mmol) of tert-butyl1-((tert-butyldimethylsilyloxy)methyl)-3-hydroxycyclobutylcarbamate(prepared by reduction ofethyl-1[[(tert-butyloxy)carbonyl]amino]-3-hydroxycyclobutane-1-carboxylateas described in J. Med. Chem., 1990 33(10) 2905-2915) and proton sponge(N,N,N′,N′ tetramethylnapthalene-1,8 diamine) (1.63 g, 6.04 mmol) weredissolved in DCM (18 mL), cooled down to 0° C., and 447 mg (3.02 mmol)of trimethyloxonium borontetrafluoride was added in one portion as asolid under vigorous stirring. The reaction mixture was stirred for 3 hand diluted with DCM (50 mL) and brine (20 mL), added under vigorousstirring. The organic phase was washed with sodium bicarbonate, brine,dried over sodium sulphate, evaporated and purified on short silicacolumn (DCM as an eluent). The resulting product was dissolved in THF (5mL), and a solution of tetrabutylammonium fluoride in THF (1M, 4.5 mL)was added, and the reaction was stirred at room temperature for 4.5 h.The reaction was monitored by TLC and once deemed to have reachedcompletion, it was absorbed onto silica and purified on silica(EtOAc-hexane 1:1 to neat EtOAc) to give the title compound (251 mg,72%). LC/MS 232 (M+1).

Step b) tert-Butyl 1-formyl-3-methoxycyclobutylcarbamate (BB4-b)

Alcohol BB4-a was dissolved in DCM (20 mL) and Dess-Martin reagent wasadded in one portion. The reaction was stirred for 2.5 hours. Once thereaction was deemed to have reached completion, it was diluted with 50mL of DCM and 20 mL of 10% Na₂S₂O₃ was added. The mixture was stirred,washed with sodium bicarbonate, brine, and the organic phase was driedover sodium sulphate. Purification on silica (EtOAc-hexane 1:1 to neatEtOAc) gave the title compound (500 mg, 59%).

Step c) tert-Butyl1-(2-(cyclopropylamino)-1-hydroxy-2-oxoethyl)-3-methoxycyclobutylcarbamate(BB4)

Aldehyde BB4-b 498 mg (1.56 mmol) was dissolved in dry DCM (8 mL).Pyridine (0.52 mL) was added under stirring conditions, followed byadding cyclopropyl isonitrile. The reaction was placed in an ice-bathand TFA (0.25 mL) was added dropwise during 20 min. The reaction mixturewas stirred overnight. The reaction was then deemed to have reachedcompletion and was washed with 1 M HCl, sodium bicarbonate, brine, andthe organic phase was dried over sodium sulphate and evaporated. Theremaining residue was dissolved in dioxane and stirred with lithiumhydroxide solution overnight and then neutralized with citric acid. Theproduct was extracted with EtOAc from the resulting solution andpurified on silica (EtOAc-hexane 1:3 to 1:1) which gave 263 mg of thetitle compound (54%) LC/MS 314 (M+1).

Building Block 5 (BB5)—a P2 Building Block

Step a) 2-tert-Butoxycarbonylamino-3-chloro-propionic acid methyl ester(BB5-a)

A solution of triphenylphosphine (65.8 g, 0.251 mol)) andhexachloroethane (59.4 g, 0.251 mol) in dichloromethane (850 mL) wasadded in one portion to a solution of N-Boc-serine methyl ester (50 g,0.228 mol) in dichloromethane (170 mL) under argon atmosphere. Thereaction mixture was stirred at room temperature for 2 h and then thereaction was quenched with a saturated solution of NaHCO₃ (150 mL). Theorganic product was extracted with dichloromethane (2×300 mL) and thecombined organic layers was washed with brine (300 mL) and dried overanhydrous sodium sulphate. The solution was concentrated under reducedpressure and then triturated with Et₂O (500 mL). After filtration andevaporation of the solvent, the crude product was purified bychromatography on a silica column eluted with 6-8% EtOAc in pet ether)which gave the title compound (42 g, 78%).

Step b) (2-Chloro-1-hydroxymethyl-ethyl)-carbamic acid tert-butyl ester(BB5-b)

Lithium borohydride (4.3 g, 0.195 mol) was added in portions to astirred solution of BB5-a (42 g, 0.177 mol) in EtOH-THF 9:1 at 0° C.under argon atmosphere. The reaction was stirred for 8 h at roomtemperature then quenched with a saturated solution of ammonium chloride(20 mL). The product was extracted with EtOAc (2×300 mL). The combinedorganic layers was washed with brine (300 mL) and dried over anhydroussodium sulphate. The solvent was evaporated under reduced pressure andthe afforded crude product was purified by chromatography on a silicacolumn eluted with 15% EtOAc in pet ether, which gave the title compound(27.5 g, 74%).

Stepc)[2-Chloro-1-(2-trimethylsilanyl-ethoxymethoxymethyl)-ethyl]-carbamicacid tert-butyl ester (BB5-c)

(2-Chloromethoxy-ethyl)-trimethyl-silane (26.18 g, 0.157 mol) was addeddrop-wise to a stirred solution of compound BB5-b (27.5 g, 0.131 mol)and N,N-diisopropylethylamine (27.4 mL, 0.157 mol) in dichloromethane(350 mL) at 0° C. under argon atmosphere. The reaction was allowed toattain room temperature and stirred for 18 h. The reaction mixture wasconcentrated under vacuum and then diluted with EtOAc (150 mL). Theproduct was extracted with EtOAc (2×200 mL) and the organic layer waswashed with brine (100 mL) and dried over anhydrous sodium sulphate. Thesolvent was evaporated under reduced pressure and the afforded crudeproduct was purified by chromatography on silica a column eluted with 5%EtOAc in pet ether, which gave the title compound (25.5 g, 57%).

Stepd)[2-(1-Hydroxy-cyclobutyl)-1-(2-trimethylsilanyl-ethoxymethoxymethyl)-ethyl]-carbamicacid tert-butyl ester (BB5-d)

n-BuLi (10 mL, 0.016 mol, 1.6 M solution in hexanes) was added drop-wiseto a stirred solution of compound BB5-c (2 g, 5.88 mmol) in THF (170 mL)at −78° C. under argon atmosphere. The stirring was continued for 15min, followed by drop-wise addition of LiNp (104 mL, 0.42 M solution inTHF, 0.044 mol) over 5 min. The dark solution was stirred at −78° C. for1 h and then cyclobutanone (0.88 mL, 11.77 mmol) was added drop-wise.The reaction mixture was stirred at −78° C. for 16 h then quenched witha saturated solution of NH₄Cl (50 mL) and allowed to warm to roomtemperature. The reaction was diluted with ether (100 mL) and asaturated solution of NH₄Cl (10 mL). The layers were separated and theaqueous layer was extracted with ether (2×100 mL). The combined organiclayers was dried (Na₂SO₄) and the solvent was evaporated under reducedpressure. The crude product was purified by chromatography on a silicacolumn eluted with heptane:ether 3:2, which gave the title compound(1.54 g, 70%,).

Step e)[2-(1-Fluoro-cyclobutyl)-1-(2-trimethylsilanyl-ethoxymethoxymethyl)-ethyl]-carbamicacid tert-butyl ester (BB5-e)

BB5-d (0.5 g, 1.33 mmol), 50% Deoxofluor in THF (excess) and pyridine(excess) were mixed in DCM (10 mL). The resulting mixture was stirred atrt over night. The reaction mixture was washed with 10% citric acid (aq)and sat. NaHCO₃ (aq). The organic phase was dried (Na₂SO₄) andevaporated. The afforded crude product was purified by chromatography ona silica column using hexane:EtOAc (7:1 to 2:1) as eluent, which gavethe title compound (192 mg, 38%).

Step f) [2-(1-Fluoro-cyclobutyl)-1-hydroxymethyl-ethyl]-carbamic acidtert-butyl ester (BB5-f)

A solution of BB5-e (192 mg, 0.51 mmol) in 0.1 M HCl in MeOH (20 mL) wasstirred for 3 hours, then triethylamine (1 mL) was added and thesolution was concentrated. The afforded crude product was purified bychromatography on a silica column using hexane:EtOAc (2:1) as eluent,which gave the title compound (69.3 mg, 55%) as a white solid.

Step g) 2-tert-Butoxycarbonylamino-3-(1-fluoro-cyclobutyl)-propionicacid (BB5)

BB5-f (69 mg, 0.279 mmol) and pyridine dichromate (1.15 g, 3.05 mmol)were dissolved in dry DMF (5 mL). After five hours H₂O (15 mL) was addedand the product was extracted with DCM (3×20 mL). The combined organiclayers was dried (Na₂SO₄) and evaporated. The crude was purified bychromatography on a silica column using hexane:EtOAc (1:1) followed byEtOH (100%) as eluent. This afforded the title compound as a white solid(22.3 mg, 31%), 262.4 [M+H]⁺.

Building Block 6 (BB6)—a P1-Prime Side Building Block

Step a) Acetoxy-(1-tert-butoxycarbonylamino-cyclobutyl)-acetic acidBB6-a

(1-Tert-butoxycarbonylaminocyclobutyl)-hydroxyacetic acid (201.5 mg,0.822 mmol) was stirred with acetic anhydride (95 mL) in pyridine (1.5mL) for 24 h. The mixture was first concentrated under vacuum, and thendiluted with 5 mL EtOAc. The organic solution was washed with 1N HCl (2mL) followed by saturated aqueous NaCl (2 mL), dried (Na₂SO₄), andevaporated under vacuum which gave the title compound. LC-UV/MS API-ES−m/z 286 [M−H]⁻

¹H NMR (400 MHz, DMSO-d6) δ ppm 7.11 (s, 1H), 5.00 (s, 1H, CHOAc), 2.28(m, 2H), 2.07 (s, 3H, Me), 2.04 (m, 2H), 1.81 (m, 1H), 1.67 (m, 1H),1.35 (s, 9H, Boc).

Step b) Acetic acid(1-tert-butoxycarbonylamino-cyclobutyl)-(4-methyl-thiazol-2-ylcarbamoyl)-methylester (BB6-b)

A mixture of the α-acetoxy carboxylic acid BB6-a (0.23 mmol) and1,1′-carbodiimidazole (87 mg, 0.54 mmol) in dry THF (5.6 mL) was stirredat rt. After 18 h, 4-methyl-2-aminothiazole hydrochloride (0.28 mmol),imidazole (20 mg, 0.29 mmol), and DMAP (0.5 mg) were added and stirringwas continued at rt. After 26 h, very little amide was formed, so themixture was heated at 65° C. for 4 h, and then concentrated undervacuum. The residue was partitioned between 10 mL EtOAc and 10 mLsaturated aqueous NaCl. The aqueous phase was extracted further withEtOAc (2×10 mL). The organic phases were combined, dried (Na₂SO₄), andevaporated in vacuo to give 152 mg oil as crude material. Flash columnchromatography (silica, 80/20 CH₂Cl₂—acetone) gave the title compound aswhite solids (62.8 mg, 71% yield).

TLC Rf (9/1 CH₂Cl₂—acetone) 0.70, LC-UV/MS 97% DAD, API-ES+ m/z 384[M+H]⁺

¹H NMR (400 MHz, DMSO-d6) δ ppm 6.95 (s, 1H), 6.76 (s, 1H, thiazole),5.16 (s, 1H, CHOAc), 2.26 (s, 3H, thiazole Me), 2.14 (s, 3H, Ac),2.4-2.1 (m, 4H), 1.8-1.6 (m, 2H), 1.33 (m, 9H).

Step c){1-[Hydroxy-(4-methyl-thiazol-2-ylcarbamoyl)-methyl]-cyclobutyl}-carbamicacid tert-butyl ester (BB6)

The acetyl group was hydrolyzed by stirring BB6-b (56 mg, 0.15 mmol)with aqueous LiOH (1N, 0.30 mL) in 1 mL methanol for 1 h at rt. Thereaction mixture was concentrated, and then partitioned between 5 mleach EtOAc and saturated aqueous NaCl. The aqueous phase was extractedfurther with EtOAc (2×5 mL). The organic phases were combined, dried(Na₂SO₄), and evaporated in vacuo to give 39.6 mg solids as crudematerial. Flash column chromatography (silica, 95/5 CH₂Cl₂—acetone) gavethe title compound as white solids (36.2 mg, 71% yield). LC-UV/MS 100%DAD, AP-ES+ m/z 342 [M+H]⁺

Building Block 7 (BB7)—a P1 Building Block

Step a) Tert-butyl3-fluoro-1-(1-hydroxy-2-(methylamino)-2-oxoethyl)cyclobutylcarbamate(BB7)

Potassium carbonate (147.5 mg, 1.06 mmol) was added to a solution of theP1-building block BB3 (254 mg, 0.96 mmol) in DMF (5 mL) followed byaddition of methyl iodide (72 μL, 1.15 mmol). The reaction mixture wasstirred at room temperature for 2.5 h and then partitioned between DCMand aq. NaHCO₃ (sat.). The phases were separated and the organic layerwas washed with water, dried (Na₂SO₄) and concentrated. The cruderesidue of the formed methyl ester was dissolved in a solution ofmethylamine in ethanol (33% (w/w), 10 mL), heated at 60° C. for 2 daysand then concentrated to give the α-hydroxy amide BB7 which was pureenough to be used in the next step without further purification. MS m/z277.4 (M+H)⁺.

Building Block 8—a P1-Prime Side Building Block (BB8)

Step a)[1-(tert-Butylcarbamoyl-hydroxy-methyl)-3-methoxy-cyclobutyl]-carbamicacid tert-butyl ester (BB8-a)

Aldehyde BB4-b, 4.45 mmol, was reacted according to the procedure asdescribed for the preparation of BB4-c, but using tert-butyl isocyanide(6.68 mmol) instead of cyclopropyl isonitrile, which gave the titlecompound (850 mg, 58%), (TLC: rf=0.61 (EtOAc:hexane 1:1).

Step d) (1-tert-Butoxycarbonylamino-3-methoxy-cyclobutyl)-hydroxy-aceticacid (BB8-b)

Compound DJ14 (850 mg, 2.57 mmol) was refluxed with 6N HCl (60 mL) for16 h until the amide hydrolysis was complete. The solvent was evaporatedunder reduced pressure and co-evaporated with water. The product wasdissolved in THF:H₂O (7:3 v/v, 50 mL), cooled to 0° C. and Et₃N (1.4 mL,10.2 mmol) was added followed by di-tert-butyl dicarbonate (2.25 g, 10.2mol). The mixture was stirred at room temperature overnight. Thereaction mixture was washed with EtOAc followed by acidifying to pH3with 1N HCl and extracted with EtOAc (2×50 mL). The organic layer waswashed with brine and dried over anhydrous Na₂SO₄. The solvent wasevaporated under reduced pressure which afforded the title P1 buildingblock as a solid (360 mg, yield 51%).

Step e){1-[Hydroxy-(1-methyl-1H-pyrazol-3-ylcarbamoyl)-methyl]-3-methoxy-cyclobutyl}-carbamicacid tert-butyl ester (BB8)

The hydroxy acid BB8-b was reacted with 1-methyl-1H-pyrazol-3-amineaccording to the procedure described in Example 1, step a, which gavethe title compound (232 mg, yield 50%).

Building Block 9—a P1-Prime Side Building Block (BB9)

Tert-butyl(1R,3S)-3-fluoro-1-((S)-1-hydroxy-2-(1-D₃-methyl-1H-pyrazol-3-ylamino)-2-oxoethyl)cyclobutylcarbamate(BB9)

D₃-methyliodide (4.42 mmol, 0.281 mL) was added to a stirred solution of3-nitropyrazole (4.42 mmol, 500 mg) and DBU (0.75 mL) in DMF. After 16h, aq. NaHCO₃ was added and the mixture was extracted with DCM. Theorganic phase was carefully concentrated and the afforded crude productwas purified by column chromatography on silica gel eluted with DCM. Theafforded residue was dissolved in MeOH, hydrogenated over Pd/C-10%,filtered through Celite and concentrated, which gave1-(D₃-methyl)-1H-pyrazol-3-ylamine. 1-(D₃-methyl)-1H-pyrazol-3-ylaminewas the reacted with BB3 according to the procedure described in Example1, step a, which gave the title compound.

Example A1

Step a){3-Fluoro-1-[hydroxy-(1-methyl-1H-pyrazol-3-ylcarbamoyl)-methyl]-cyclobutyl}-carbamicacid tert-butyl ester (A1-a)

1-Methyl-1H-pyrazol-3-amine (1 eq.) and DIEA (4 eq) was added to asolution of the P1-building block BB3 (1 eq) dissolved in DMF. Thesolution was cooled to 0° C. and after 10 minutes HATU (1 eq) was added.After approximately 2 hours at RT, LC-MS showed product and no startingmaterial and the solvent was removed by rotary evaporation. The crudeproduct was dissolved in 40 mL EtOAc and washed with 25 mL sat.NaHCO_(3 (aq)). The organic phase was dried with Na₂SO₄, filtered andevaporated to dryness. The crude product was purified on a 25 g silicacolumn on a Biotage Flashmaster, which gave the title product.

Step b)2-(1-Amino-3-fluoro-cyclobutyl)-2-hydroxy-N-(1-methyl-1H-pyrazol-3-yl)-acetamide(A1-b)

Compound A1-a (363 mg, 1.06 mmol) was treated with a 7.5 mL of a mixtureof TFA:DCM:TIS:water (20:80:1:1). After 2 h at room temperature, LC/MSanalysis confirmed complete removal of the Boc group and the solutionwas concentrated in vacuo and azeotroped with dichloromethane (3×) toremove excess TFA. The crude product (339 mg, 0.9 mmol) was dissolved inDCM (10 mL) and added to a solution of2-amino-3-(1-methyl-cyclopentyl)-propionamide (prepared as described inEx. 1 of WO2006/064286) (1.2 eq), PyBOP (1.2 eq) anddiisopropylethylamine (4 eq) in DCM (5 mL) that had been stirred at roomtemperature for 5 min. The mixture was stirred at room temperature untilLC/MS analysis indicated complete amide formation (˜30 min) DCM wasadded to the reaction and the organic phase was washed with 0.1 M HCl(aq) (2×) and 10% NaHCO₃ (aq) (2×). The organic phase was dried andconcentrated in vacuo to afford the title compound which was used insubsequent step without further purification.

Step c)N-{3-Fluoro-1-[hydroxy-(1-methyl-1H-pyrazol-3-ylcarbamoyl)-methyl]-cyclobutyl}-3-(1-methyl-cyclopentyl)-2-propionylamino-propionamide(A1-c)

Compound A1-b (0.9 mmol) was treated with a 7.5 mL of a mixture ofTFA:DCM:TIS:water (20:80:1:1). After 2 h at room temperature, LC/MSanalysis confirmed complete removal of the BOC group and the solutionwas concentrated in vacuo and azeotroped with dichloromethane (3×) toremove excess TFA. The residue was dissolved in DCM and extracted with0.1M HCl (aq) (3×). The pooled aqueous layers were basified to pH 10with NaOH (aq) and the product extracted with DCM (3×). The pooledorganic layers were dried (MgSO₄) and concentrated in vacuo. Theafforded crude compound (33 mg, 83 mmol) was dissolved in DCM (2 mL) andDIEA (3 eq.) and propionylchloride (91.3 mmol) was added. The reactionwas stirred at room temperature for 1 h after which time LC/MS analysisindicated complete acylation. The reaction was quenched by addition ofMethanol (0.5 mL) and the reaction was concentrated in vacuo. Theresidue was dissolved in DCM (5 mL) and the organic phase was washedwith 0.1M HCl (aq) (2×) and 10% NaHOC₃ (aq) (2×), eluted through ahydrophobic Phase separator and concentrated in vacuo, which gave thetitle compound.

Step d)N-[3-Fluoro-1-(1-methyl-1H-pyrazol-3-ylaminooxalyl)-cyclobutyl]-3-(1-methyl-cyclopentyl)-2-propionylamino-propionamide(A1)

The residue afforded in step c was re-dissolved in DCM (1.5 mL) andDess-Martin periodinane (91.3 mmol) was added in one portion at roomtemperature. The reaction was stirred at room temperature for 2 h afterwhich time LC/MS analysis indicated complete oxidation. The reactionmixture was diluted with DCM and the solution washed with a 1:1 mixtureof 10% Na₂S₂O₃ (aq) and 10% NaHCO₃ (aq). The organic layer was elutedthrough a hydrophobic Phase Separator and concentrated in vacuo. Theresidue was purified by preparative LC/MS to afford the target compound(Yield: 47%, LC/MS: t_(R)=4.55 min, 450.2 [M+H]⁺)

Examples A2-A10

The compounds illustrated in the table below were prepared analogouslyto the procedure outlined in Example 1 using the appropriateR^(1a)R^(1b) amines, P1/P2 and P3 building blocks acid, followed by DessMartin oxidation to the end product α-keto amide.

TABLE A1

Ex. R⁴ R³ R^(2a) R^(2b) R^(1b) [M + H]⁺ A2 methyl1-methylcyclopentylmethyl F H 1-methylpyrazol-3-yl 436.3 A3 t.butyl1-methylcyclopentylmethyl F H 1-methylpyrazol-3-yl 478.14 A4¹ ethyl1-fluorocyclopentylmethyl F H 1-methylpyrazol-3-yl 453.96 A5¹ t.butyl1-fluorocyclopentylmethyl F H 1-methylpyrazol-3-yl 481.98 A6^(2,3)methyl 1-methylcyclopentylmethyl H H 1-methylpyrazol-3-yl 418.2 A7t.butyl 1-fluorocyclopentylmethyl F F 1-methylpyrazol-3-yl 500.2A8^(4,5) methyl 1-methylcyclopentylmethyl F H 1-cyclopropylmethyl- 504pyrazol-3-yl A9^(4,5) methyl 1-methylcyclopentylmethyl F H1-(2,2,2-trifluoroethyl)- 476 pyrazol-3-yl A10² methyl1-fluorocyclobutylmethyl F F 1-methylpyrazol-3-yl 444.16 ¹Removal of theBoc group in step b was performed using 4 M HCl in dioxane and/or MeOH.²Removal of the Boc group in steps b and c was performed using 4 M HClin dioxane and/or MeOH. ³The P2 building block was coupled to theP1-P1′-building block using DMF as solvent and HATU as coupling reagent.⁴The P1′ amine was coupled to the P1-building block using HATU ascoupling agent. ⁵Removal of the Boc group in steps b and c was performedusing MeOH:AcCl 9:1.

Example A11

Step a){3,3-Difluoro-1-[hydroxy-(1-methyl-1H-pyrazol-3-ylcarbamoyl)-methyl]-cyclobutyl}-carbamicacid tert-butyl ester (A11-a)

1-Methyl-1H-pyrazol-3-amine (158 mg, 1.63 mmol) and DIEA (1.08 mL, 6.52mmol) was added to a solution of(1-tert-Butoxycarbonylamino-3,3-difluoro-cyclobutyl)-hydroxy-acetic acid(1.63 mmol) dissolved in DMF (20 mL). The solution was cooled to 0° C.and after 10 minutes HATU (620 mg, 1.63 mmol) was added. Afterapproximately 2 hours at RT, LC-MS showed product and no startingmaterial and the solvent was removed by rotary evaporation. The crudeproduct was dissolved in 40 mL EtOAc and washed with 25 mL sat.NaHCO_(3 (aq)). The organic phase was dried with Na₂SO₄, filtered andevaporated to dryness. The crude product was purified on a 25 g silicacolumn on a Biotage Flashmaster, which gave the title product as a whitesolid (92%). TLC rf: 0.07 in heptane:ethyl acetate 1:1.

Step b)[1-{3,3-Difluoro-1-[hydroxy-(1-methyl-1H-pyrazol-3-ylcarbamoyl)-methyl]-cyclobutylcarbamoyl}-2-(1-methyl-cyclopentyl)-ethyl]-carbamicacid tert-butyl ester (A11-b)

A11-a (0.629 mmol) was dissolved in MeOH (3 mL). A solution of HCl indioxane (4M, 1.5 mL) was added. The solution was stirred at RT for 16 h,then concentrated and co-evaporated with toluene. The afforded residue(0.63 mmol) was dissolved in anhydrous DMF (1 mL) and then added at 0°C. to a cold solution of 2-amino-3-(1-methyl-cyclopentyl)-propionamide(prepared as described in Ex. 1 of WO2006/064286) (187.5 mg, 0.69 mmol)and HATU (263 mg, 0.69 mmol) in dry DMF (2 mL). DIEA (550 μL, 3.15 mmol)was added, and the reaction mixture was stirred at 0° C. for 30 minutes,then at RT for 2 h. The solution was concentrated to vacuum, the residuewas dissolved in DCM (3 mL) and applied to a silica column (10 g). Thecompound was purified by flash chromatography (heptane: ethyl acetate75:25-25:75) which gave the title compound (90%) as two chromatographicpeaks. MS m/z 478.2 (M+H)⁺.

Step c)[1-[3,3-Difluoro-1-(1-methyl-1H-pyrazol-3-ylaminooxalyl)-cyclobutylcarbamoyl]-2-(1-methyl-cyclopentyl)-ethyl]-carbamicacid tert-butyl ester (A11-c)

The α-hydroxy amide A11-b was oxidized according to the method describedin Example A1 step d. Purification by prep LCMS (50-70% gradient, mobilephase: acetonitrile-water, 1% NH₄OH) gave the title compound. MS m/z476.2 (M+H)⁺. Purity assessed by analytical LCMS 99.4%.

Step d)2-Acetylamino-N-[3,3-difluoro-1-(1-methyl-1H-pyrazol-3-ylaminooxalyl)-cyclobutyl]-3-(1-methyl-cyclopentyl)-propionamide(A11)

Carbamate A11-c (196 mg, 0.383 mmol) was dissolved in a pre maid mixtureof methanol and acetyl chloride 9:1 (10 mL). The mixture was stirred for6 hrs whereafter the solvent was removed and the crude product driedover night under high vacuum. The crude product was dissolved in DMF (10mL) and AcCl (27 μL, 0.383 mmol) was added followed by DIEA (253 μL,1.53 mmol). After approximately 2 hours at RT, LC-MS showed product andno starting material and the solvent was removed by rotary evaporation.The crude product was dissolved in 40 mL of EtOAc and washed with 25 mLof sat. NaHCO_(3 (aq)). The organic phase was dried with Na₂SO₄,filtered and evaporated on silica. The crude product was purified on apre packed 10 g silica column using a gradient going from heptane: EtOAC1:1 towards pure EtOAc, towards pure acetone, which gave the titlecompound (109 mg, 63%) yield. [M+H]⁺454.

The compounds illustrated in the table below were prepared according tothe procedure outlined in Example A11, using the appropriateR^(1a)R^(1b)amine, P1-, P2-building blocks, and acid chloride.

TABLE A2

Ex. R⁴ R³ R^(2a) R^(2b) R^(1b) [M + H]⁺ A12 methyl2-fluoro-2-methylbutyl F F 1-methylpyrazol-3-yl 432 A13 methyl1-methylcyclobutylmethyl F F 1-methylpyrazol-3-yl 440 A14 methylcyclohexylmethyl F F 1-methylpyrazol-3-yl 454 A15 methyl homo-t-butyl FF 1-methylpyrazol-3-yl 428 A16² methyl 1-methylcyclopentylmethyl OMe Hcyclopropyl 408 A17 methyl 1-fluorocyclopentylmethyl F H1-methylpyrazol-3-yl 440 A18 methyl 1-methylcyclobutylmethyl F H1-methylpyrazol-3-yl 422 A19¹ chloromethyl 1-methylcyclopentylmethyl OMeH cyclopropyl 442 A20² methyl 1-methylcyclopentylmethyl F F4-methylthiazol-2-yl 435 A21² methyl 1-fluorolcyclopentylmethyl F F1-methylpyrazol-3-yl 458 A22 methyl 1-methylcyclobutylmethyl F Hcyclopropyl 382 A23 methyl 1-fluorolcyclopentylmethyl F H cyclopropyl396 A24 methyl 1-methylcyclopentylmethyl F H cyclopropyl 400 A25^(2,3)isopropyl 1-methylcyclopentylmethyl F F 1-methylpyrazol-3-yl 482 A26⁴ethyl 1-methylcyclopentylmethyl F F 1-methylpyrazol-3-yl 467.3 A27⁴chloroethyl 1-methylcyclopentylmethyl F F 1-methylpyrazol-3-yl 487.2¹The stereochemistry at the steric centre to which R^(2a) and R^(2b) isattached is not defined. ²The Boc group of the carbamate was removedusing 2M HCl in dioxane. ³The P3 moiety was introduced using theappropriate acid anhydride. ⁴The P3 moiety was introduced in a HATUpromoted coupling with the appropriate acid.

Example A28

Step a) Acetyl-cyclohexylglycine (A28-a)

L-Boc-cyclohexylglycine (0.500 g, 1.94 mmol) was dissolved in DCM (5 mL)and cooled to 0° C. TFA (5 mL) was added and the reaction was stirredfor 30 min. The mixture was concentrated in vacuo and the residue wasresuspended in a few mL of toluene and the mixture evaporated by rotaryevaporation (process repeated thrice) followed by drying under vacuum.The crude TFA salt was dissolved in H₂O (6 mL), cooled to 0° C. and NaOH(776 mg, 19.4 mmol) was added. Stirring was continued until ahomogeneous solution was achieved. Acetic anhydride (3.88 mmol, 0.388mL) was added and the reaction was stirred for 2 h. The mixture wasdiluted with 1 M NaOH (25 mL) and the resulting solution was washed withEt₂O, then acidified with 3 M HCl to pH=1, and extracted with EtOAc(3×20 mL). The combined organic phases were dried (MgSO₄), filtered andevaporated to give 120 mg of the title compound in high purity. LC-MSES+200.2 [M+H]⁺.

Step b)2-[1-(2-Acetylamino-2-cyclohexyl-acetylamino)-cyclobutyl]-2-hydroxy-N-(1-methyl-1H-pyrazol-3-yl)-acetamide(A28-b)

2-(1-Amino-cyclobutyl)-2-hydroxy-N-(1-methyl-1H-pyrazol-3-yl)-acetamide(prepared according to the method described in Example 1, step a (50 mg,0.154 mmol) was treated with 2 mL 4 M HCl in dioxane to yield thedihydrochloric salt of the free amine after freeze-drying. This wasreacted with compound A28-a according to the coupling proceduredescribed in Example C9 step b and the product was purified by gradientcolumn chromatography (1-10% MeOH in DCM) to give the title compound (30mg, 48%) LC-MS ES+406.3 [M+H]⁺.

Step c)2-[1-(2-Acetylamino-2-cyclohexyl-acetylamino)-cyclobutyl]-N-(1-methyl-1H-pyrazol-3-yl)-2-oxo-acetamide(A28)

Compound A28-b was oxidized according to Example 1 step d and theproduct was purified by preparative HPLC on a Phenomenex Gemini-NXcolumn Eluents: A 0.01M NH₃ in H₂O; B 45% MeCN, 45% THF, 10% H₂O,gradient 15-28% B. This gave 15 mg of the title compound (50% yield)after lyophilisation. Purity >99% (HPLC-DAD), LC-MS ES+403.1.

Example A29

2-Acetylamino-3-(1-fluoro-cyclopentyl)-N-[1-(1-methyl-1H-pyrazol-3-ylaminooxalyl)-cyclobutyl]-propionamide(A29)

The title compound was prepared according to the method described inExample 1, using the appropriate building blocks. Removal of the Bocgroup in steps b and c was performed by treatment with 4M HCl indioxane/MeOH. Yield: 16%, LC/MS: t_(R)=4.0 min, 421.2 [M+H]⁺).

Example U1

Step a)3-(1-Methyl-cyclopentyl)-2-[(pyrrolidine-1-carbonyl)-amino]-propionicacid lithium salt (U1-a)

(S)-2-(tert-Butoxycarbonylamino)-3-(1-methylcyclopentyl)propanoic acid,(prepared as described in Ex. 1 of WO2006/064286) (272 mg, 1 mmol) wasdissolved in MeOH (1 mL). 4M

HCl in dioxane was added dropwise (3 mL) at room temperature. Afterapproximately 3 hours, the solvent was removed by rotary evaporation andco-evaporated with MeOH (2×) to remove excess HCl. The afforded crudecompound was used in the following step without further purification.

Step b) 2-(3,3-Diethyl-ureido)-3-(1-methyl-cyclopentyl)-propionic acidlithium salt (U1-b)

Compound U1-a (19 mg, 86 mmol) was dissolved in THF (1 mL) andtriethylamine (3 eq) and diethylcarbamoyl chloride (1 eq) were added.The reaction was heated to 50° C. in a sealed tube for 16 h. LC/MSanalysis showed 90% conversion. EtOAc was added to the reaction solutionand the organic phase was washed with 01M HCl (aq) (3×). The organiclayer was dried (MgSO₄), filtered and the solvent removed in vacuo. Theresulting crude methyl ester was dissolved in THF (mL), 1M LiOH inmethanol (3 eq) was added, and the solution stirred at room temperaturefor 16 h. LC/MS analysis indicated complete ester hydrolysis and thesolvent was removed in vacuo to afford the lithium salt which was usedin subsequent step without further purification.

Step c)2-(3,3-Diethyl-ureido)-N-{3-fluoro-1-[hydroxy-(1-methyl-1H-pyrazol-3-ylcarbamoyl)-methyl]-cyclobutyl}-3-(1-methyl-cyclopentyl)-propionamide(U1-c)

Compound 1a was treated with a 7.5 mL of a mixture of TFA:DCM:TIS:water(20:80:1:1). After 2 h at room temperature, LC/MS analysis confirmedcomplete removal of the BOC group and the solution was concentrated invacuo and azeotroped with dichloromethane (3×) to remove excess TFA. Theafforded crude TFA salt (64 mmol) was dissolved in DCM (2 mL) and addedto a solution of U1-b (1.2 eq) PyBOP (1.2 eq) in DCM (2 mL) that hadbeen pre-stirred at room temperature for 10 min. The mixture was stirredat room temperature for 16 h after which time the reaction wasinterrupted. DCM was added to the reaction and the organic phase waswashed with 0.1 M HCl (aq) (2×) and 10% NaHCO₃ (aq) (2×). The organicphase was dried and concentrated in vacuo and the residue purified bypreparative LC/MS.

Step d)2-(3,3-Diethyl-ureido)-N-[3-fluoro-1-(1-methyl-1H-pyrazol-3-ylaminooxalyl)-cyclobutyl]-3-(1-methyl-cyclopentyl)-propionamide(U1)

The alcohol U1-c was dissolved in DCM (1.5 mL) and Dess-Martinperiodinane (1.5 eq) was added in one portion at room temperature. Thereaction was stirred at room temperature for 2 h after which time LC/MSanalysis indicated complete oxidation. The reaction was diluted with DCMand the solution washed with a 1:1 mixture of 10% Na₂S₂O₃ (aq) and 10%NaHCO₃ (aq). The organic layer was eluted through a hydrophobic PhaseSeparator and concentrated in vacuo. The residue was purified bypreparative LC/MS which gave the title compound. (Yield: 4.2 mg, LC/MS:t_(R)=5.5 min, 493.16 [M+H]⁺).

Examples A30-A38

The compounds illustrated in the table below were prepared analogouslyto the procedure outlined in Example 1 using the appropriateR^(1a)R^(1b), amine, P1- and P2-building blocks, and acid chlorides,followed by Dess Martin oxidation to the end product α-keto amide.

TABLE A3

Ex. R⁴ R³ R^(2a) R^(2b) R^(1b) [M + H]⁺ A30^(2,3) isopropyl1-fluorocyclopentylmethyl F H 1-methylpyrazol-3-yl 468.0 A31^(2,3,6)tert.butyl 1-fluorocyclopentylmethyl OMe H 1-methylpyrazol-3-yl 494.4A32^(3,4) tert.butyl 1-fluorocyclopentylmethyl H H 1-methylpyrazol-3-yl464.1 A33² methyl cyclohexylethyl H H 1-methylpyrazol-3-yl 432.36A34^(2,3,7) 2,2,2-tri- 1-fluorocyclopentylmethyl F F1-methylpyrazol-3-yl 526.01 fluoro- ethyl A35^(2,3,7) difluoro-1-methylcyclopentyl- F F 1-methylpyrazol-3-yl 490.29 methyl methylA36^(2,3,7) difluoro- 1-fluorocyclopentylmethyl F F 1-methylpyrazol-3-yl494.29 methyl A37^(1,2,7) D₉-t.butyl 1-fluorocyclopentylmethyl F F1-methylpyrazol-3-yl 508.9 A38^(5,7) sec. butyl 1-methylcyclopentyl- H H1-methylpyrazol-3-yl 460 methyl ¹Removal of the Boc group in step b wasperformed using 4 M HCl in dioxane and/or MeOH. ²Removal of the Bocgroup in steps b and c was performed using 4 M HCl in dioxane and/orMeOH. ³The P2 building block was coupled to the P1-P1′-building blockusing DMF as solvent and HATU as coupling reagent. ⁴The P1′ amine wascoupled to the P1-building block using HATU as coupling agent. ⁵Removalof the Boc group in steps b and c was performed using MeOH:AcCl 9:1.⁶The isomeric ratio of the OMe in R^(2a)/R^(2b) is 1:1. ⁷In step c, theP3 building block was introduced in a HATU-promoted coupling with theappropriate acid.

Example U2

Step a)3-(1-Fluoro-cyclopentyl)-N-[3-fluoro-1-(1-methyl-1H-pyrazol-3-ylaminooxalyl)-cyclobutyl]-2-(3-isopropyl-ureido)-propionamide(U2)

The α-hydroxy amide precursor of carbamate C18 was treated with 4M HClin dioxane for 0.5 h and then lyophilised. The thus afforded HCl-saltwas treated with DIEA (2 eq.) and isopropyl isocyanate (1.1 eq.) inCH₂Cl₂. After 0.5 h, LC/MS analysis indicated complete turnover and thereaction mixture was diluted with CH₂Cl₂, and the organic layer waswashed with 10% citric acid (2×) and 10% NaHCO3 (2×), dried through ahydrophobic frit and concentrated in vacuo. The afforded crude compoundwas oxidized according to the procedure described in Example 1 step d,which gave the title compound. The residue was purified by preparativeLC/MS to afford the target compound, M/Z 483.4 [M+H]⁺.

Examples U3-U5

The compounds illustrated in the table below were prepared analogouslyto the procedure outlined in Example U2, using the appropriate α-hydroxyamide and ethyl isocyanate.

TABLE U1

Ex. R^(3*) R^(2a) R^(2b) M/Z U3 F F H 467.3 [M − H]⁻ U4 F H H 449.4 [M −H]⁻ U5 methyl F H 465.3 [M + H]⁺

Example A39

Step a) (9H-fluoren-9-yl)methyl1-(1-hydroxy-2-(1-methyl-1H-pyrazol-3-ylamino)-2-oxoethyl)cyclobutylcarbamate(A39-a)

{1-[Hydroxy-(1-methyl-1H-pyrazol-3-ylcarbamoyl)-methyl]-cyclobutyl}-carbamicacid tert-butyl ester (209 mg, 0.64 mmol) was treated with HCl (4M indioxane, 5 mL) overnight and then concentrated. The resultinghydrochloride salt was added to a solution of Fmoc-Cl (232 mg, 0.89mmol) in dioxane:water 2:3, whereafter NaHCO₃ (181 mg, 2.24 mmol) wasadded. The reaction mixture was stirred at room temperature and afterfor 40 min the solution was diluted with DCM and extracted. The organicphase was washed with brine, dried (NaSO₄) and concentrated.Purification by flash column chromatography (EtOAc/iso-Hexane, 4:6-6:4)gave the title compound as a white solid (257 mg, 90%). MS m/z 446.9(M+H)⁺.

Step b) (9H-fluoren-9-yl)methyl1-(2-(1-methyl-1H-pyrazol-3-ylamino)-2-oxoacetyl)cyclobutylcarbamate(A39-b)

Dess-Martin periodinane (668 mg, 1.57 mmol) was added to a solution ofalcohol A39-a (503 mg, 1.13 mmol) in anh. DCE (7 ml). The reactionmixture was stirred at room temperature for 30 min and then quenchedwith Na₂S₂O₃ (10% aq) and NaHCO₃ (aq, sat.). The phases were separatedand the organic layer was dried (Na₂SO₄) and concentrated yielding thetitle compound which was used in the next step without furtherpurification. MS m/z 444.9 (M+H)⁺.

Step c)(Z)-4-((2-(1-(1-(((9H-fluoren-9-yl)methoxy)carbonylamino)cyclobutyl)-2-(1-methyl-1H-pyrazol-3-ylamino)-2-oxoethylidene)hydrazinecarboxamido)methyl)cyclohexane-carboxylicacid (A39-c)

A trifluoroacetic salt of Webb's acid linker (593 mg, 1.78 mmol) wasadded to a solution of the ketone A39-b (1.12 mmol, crude) in methanol(25 mL). The reaction mixture was stirred at room temperature overnightand then concentrated. Purification by flash column chromatography(MeOH/DCM, 0:1-1:9) gave the title compound (0.61 g, 85%). MS m/z 642.9(M+H)⁺.

Step d) Resin bound(Z)-4-((2-(1-(1-(((9H-fluoren-9-yl)methoxy)carbonylamino)cyclobutyl)-2-(1-methyl-1H-pyrazol-3-ylamino)-2-oxoethylidene)hydrazinecarboxamido)methyl)cyclohexane-carboxylicacid (A39-d)

Amine resin (TG resin NovaSyn, 01-64-0043, 0.25 mmol/g, 2 g, 0.5 mmol)was added to a solution of acid A39-c (546 mg, 0.85 mmol), HBTU (760 mg,2 mmol), HOBt (306 mg, 2 mmol) and NMM (550 μL, 4 mmol) in DMF (13 mL).The slurry was agitated at room temperature overnight. The resin wasfiltered and then washed several times alternating with DMF, DCM andMeOH.

Step e) Resin bound4-(((Z)-2-(1-(1-(3-(((9H-fluoren-9-yl)methoxy)carbonylamino)-4-(1-methylcyclopentyl)-2-oxobutyl)cyclobutyl)-2-(1-methyl-1H-pyrazol-3-ylamino)-2-oxoethylidene)hydrazinecarboxamido)methyl)cyclohexanecarboxylicacid (A39-e)

Resin A39-d (0.5 mmol) was treated with a solution of 20% piperidine inDMF (30 mL) for 1 h, then filtered and washed with DMF (3×20 mL), andseveral times alternating with DCM and MeOH. The resulting amine resinsuspended in DMF was added to Fmoc-protected2-amino-3-(1-methyl-cyclopentyl)-propionic acid (294 mg, 0.75 mmol),HBTU (380 mg, 1 mmol), HOBt (153 mg, 1 mmol) and NMM (350 μL, 2 mmol).The slurry was agitated at room temperature over night. The resin wasfiltered, washed as described above, and dried under vacuum.

Step f) Resin bound(S,Z)-4-((2-(2-(1-methyl-1H-pyrazol-3-ylamino)-1-(1-(4-(1-methylcyclopentyl)-2-oxo-3-propionamidobutyl)cyclobutyl)-2-oxoethylidene)hydrazinecarboxamido)methyl)cyclohexanecarboxylicacid (A39-f)

Resin bound compound A39-e (0.05 mmol) was treated with a solution of20% piperidine in DMF (6 mL) for 1 h, filtered and washed with DMF (3×20mL), and several times alternating with DCM and MeOH. To the resultingamine resin suspended in DMF was added, ethyl carboxylic acid (15 μL,0.2 mmol), HBTU (76 mg, 0.2 mmol), HOBt (30 mg, 0.2 mmol) and NMM (55μL, 0.4 mmol). The slurry was agitated at room temperature over night.The resin was filtered, washed as described above, and dried undervacuum.

Step g)(S)—N-(4-(1-(2-(1-methyl-1H-pyrazol-3-ylamino)-2-oxoacetyl)cyclobutyl)-1-(1-methylcyclopentyl)-3-oxobutan-2-yl)propionamide(A39)

Resin bound amide A39-e (0.05 mmol) was treated with a solution ofTFA:H₂O 95:5 (4 mL) for 4 h. The resin was filtered and washed with DCM(3×5 mL) and MeOH (3×5 mL). The filtrate and the washes were pooled andconcentrated and the afforded crude product was purified by RP-LC-MS(0.1% NH₄OH in acetonitrile-0.1% aq. NH₄OH, 40-65% over 9 min). Overallyield 4%. MS m/z 431.9 (M+H)⁺.

Biological Examples Determination of Cathepsin K Proteolytic CatalyticActivity

Convenient assays for cathepsin K are carried out using humanrecombinant enzyme, such as that described in PDB.

ID BC016058 standard; mRNA; HUM; 1699 BP.DE Homo sapiens cathepsin K (pycnodysostosis), mRNA (cDNA cloneMGC:23107

RX MEDLINE; RX PUBMED; 12477932. DR RZPD; IRALp962G1234. DR SWISS-PROT;P43235;

The recombinant cathepsin K can be expressed in a variety ofcommercially available expression systems including E coli, Pichia andBaculovirus systems. The purified enzyme is activated by removal of theprosequence by conventional methods.

Standard assay conditions for the determination of kinetic constantsused a fluorogenic peptide substrate, typically H-D-Ala-Leu-Lys-AMC, andwere determined in either 100 mM Mes/Tris, pH 7.0 containing 1 mM EDTAand 10 mM 2-mercaptoethanol or 100 mMNa phosphate, imM EDTA, 0.1%PEG4000 pH 6.5 or 100 mM Na acetate, pH 5.5 containing 5 mM EDTA and 20mM cysteine, in each case optionally with 1M DTT as stabiliser. Theenzyme concentration used was 5 nM. The stock substrate solution wasprepared at 10 mM in DMSO. Screens were carried out at a fixed substrateconcentration of 60 μM and detailed kinetic studies with doublingdilutions of substrate from 250 μM. The total DMSO concentration in theassay was kept below 3%. All assays were conducted at ambienttemperature. Product fluorescence (excitation at 390 nm, emission at 460nm) was monitored with a Labsystems Fluoroskan Ascent fluorescent platereader. Product progress curves were generated over 15 minutes followinggeneration of AMC product.

Cathepsin S Ki Determination

The assay uses baculovirus-expressed human cathepsin S and theboc-Val-Leu-Lys-AMC fluorescent substrate available from Bachem in a 384well plate format, in which 7 test compounds can be tested in parallelwith a positive control comprising a known cathepsin S inhibitorcomparator.

Substrate Dilutions

280 μL/well of 12.5% DMSO are added to rows B-H of two columns of a 96deep well polypropylene plate. 70 μL/well of substrate is added to rowA. 2×250 μL/well of assay buffer (100 mM Na phosphate, 100 mM NaCl, pH6.5) is added to row A, mixed, and double diluted down the plate to rowH.

Inhibitor Dilutions

100 μL/well of assay buffer is added to columns 2-5 and 7-12 of 4 rowsof a 96 well V bottom polypropylene plate. 200 μL/well of assay bufferis added to columns 1 and 6.

The first test compound prepared in DMSO is added to column 1 of the toprow, typically at a volume to provide between 10 and 30 times theinitially determined rough K_(i). The rough K_(i) is calculated from apreliminary run in which 10 μL/well of 1 mM boc-VLK-AMC (1/10 dilutionof 10 mM stock in DMSO diluted into assay buffer) is dispensed to rows Bto H and 20 μl/well to row A of a 96 well Microfluor™ plate. 2 μl ofeach 10 mM test compound is added to a separate well on row A, columns1-10. Add 90 μl assay buffer containing 1 mM DTT and 2 nM cathepsin S toeach well of rows B-H and 180 μl to row A. Mix row A using amultichannel pipette and double dilute to row G. Mix row H and read inthe fluorescent spectrophotometer. The readings are Prism data fitted tothe competitive inhibition equation, setting S=100 μM and K_(M)=100 μMto obtain an estimate of the K_(i), up to a maximum of 100 μM.

The second test compound is added to column 6 of the top row, the thirdto column 1 of the second row etc. Add 1 μL of comparator to column 6 ofthe bottom row. Mix column 1 and double dilute to column 5. Mix column 6and double dilute to column 10. Using an 8-channel multistepping pipetteset to 5×10 μL, distribute 10 μL/well of substrate to the 384 well assayplate. Distribute the first column of the substrate dilution plate toall columns of the assay plate starting at row A. The tip spacing of themultichannel pipette will correctly skip alternate rows. Distribute thesecond column to all columns starting at row B. Using a 12-channelmultistepping pipette set to 4×10 μL, distribute 10 μL/well of inhibitorto the 384 well assay plate. Distribute the first row of the inhibitordilution plate to alternate rows of the assay plate starting at A1. Thetip spacing of the multichannel pipette will correctly skip alternatecolumns. Similarly, distribute the second, third and fourth rows toalternate rows and columns starting at A2, B1 and B2 respectively.

Mix 20 mL assay buffer and 20 μL 1M DTT. Add sufficient cathepsin S togive 2 nM final concentration.

Using the a distributor such as a Multidrop 384, add 30 μL/well to allwells of the assay plate and read in fluorescent spectrophotometer suchas an Ascent.

Fluorescent readings, (excitation and emission wavelengths 390 nm and460 nm respectively, set using bandpass filters) reflecting the extentof enzyme cleavage of the fluorescent substrate, notwithstanding theinhibitor, are linear rate fitted for each well.

Fitted rates for all wells for each inhibitor are fitted to thecompetitive inhibition equation using SigmaPlot 2000 to determine V, Kmand Ki values.

Cathepsin L Ki

The procedure above with the following amendments is used for thedetermination of Ki for cathepsin L.

The enzyme is commercially available human cathepsin L (for exampleCalbiochem). The substrate is H-D-Val-Leu-Lys-AMC available from Bahcem.The assay buffer is 100 mM sodium acetate 1 mM EDTA, pH5.5) The DMSOstock (10 mM in 100% DMSO) is diluted to 10% in assay buffer. Enzyme isprepared at 5 nM concentration in assay buffer plus 1 mM dithiothreitoljust before use. 2 μL of 10 mM inhibitor made up in 100% DMSO isdispensed into row A. 10 μL of 50 μM substrate (=1/200 dilution of 10 mMstock in DMSO, diluted in assay buffer).

Inhibition Studies

Potential inhibitors are screened using the above assay with variableconcentrations of test compound. Reactions were initiated by addition ofenzyme to buffered solutions of substrate and inhibitor. K_(i) valueswere calculated according to equation 1.

$\begin{matrix}{v_{0} = \frac{VS}{{K_{M}( {1 + \frac{I}{K_{i}}} )} + S}} & (1)\end{matrix}$

where v₀ is the velocity of the reaction, V is the maximal velocity, Sis the concentration of substrate with Michaelis constant of K_(M), andI is the concentration of inhibitor.

The inhibition of cathepsin S, cathepsin K and cathepsin L exhibited bya selection of the compounds of the invention represented as Ki valuesin nM is presented in Table 1.

TABLE 1 Example Ki Cat. S Ki Cat. K Ki Cat. L A1 0.8 850 3900 A6 0.81580 5200 A13 3.8 340 870 A15 20 1200 6500 A16 10 12000 33000 A19 5.67400 25000 A25 1.0 1100 2300 A30 1.0 720 3300 A31 1.8 340 1200 A36 31100 380 A37 3 640 1800 U1 1.2 510 860 U2 1.3 1300 2200 U4 0.63 620 2100

The compounds of formula I are thus potent inhibitors of cathepsin S andyet selective over the closely related cathepsin K and L.

Permeability

This experiment measures transport of inhibitors through the cells ofthe human gastroenteric canal. The assay uses the well known Caco-2cells with a passage number between 40 and 60.

Apical to Basolateral Transport

Generally every compound will be tested in 2-4 wells. The basolateraland the apical wells will contain 1.5 mL and 0.4 mL transport buffer(TB), respectively, and the standard concentration of the testedsubstances is 10 μM. Furthermore all test solutions and buffers willcontain 1% DMSO. Prior to the experiment the transport plates arepre-coated with culture medium containing 10% serum for 30 minutes toavoid nonspecific binding to plastic material. After 21 to 28 days inculture on filter supports, the cells are ready for permeabilityexperiments.

Transport plate no 1 comprises 3 rows of 4 wells each. Row 1 is denotedWash, row 2 “30 minutes” and row 3 “60 minutes”. Transport plate no 2comprises 3 rows of 4 wells, one denoted row 4 “90 minutes”, row 5 “120minutes and the remaining row unassigned.

The culture medium from the apical wells is removed and the inserts aretransferred to a wash row (No. 1) in a transport plate (plate no. 1) outof 2 plates without inserts, which have already been prepared with 1.5mL transport buffer (HBSS, 25 mM HEPES, pH 7.4) in rows 1 to 5. In A→Bscreening the TB in basolateral well also contains 1% Bovine SerumAlbumin

0.5 mL transport buffer (HBSS, 25 mM MES, pH 6.5) is added to theinserts and the cell monolayers equilibrated in the transport buffersystem for 30 minutes at 37° C. in a polymix shaker. After beingequilibrated to the buffer system the Transepithelial electricalresistance value (TEER) is measured in each well by an EVOM chop stickinstrument. The TEER values are usually between 400 to 1000Ω per well(depends on passage number used).

The transport buffer (TB, pH 6.5) is removed from the apical side andthe insert is transferred to the 30 minutes row (No. 2) and fresh 425 μLTB (pH 6.5), including the test substance is added to the apical (donor)well. The plates are incubated in a polymix shaker at 37° C. with a lowshaking velocity of approximately 150 to 300 rpm.

After 30 minutes incubation in row 2, the inserts are moved to newpre-warmed basolateral (receiver) wells every 30 minutes; row 3 (60minutes), 4 (90 minutes) and 5 (120 minutes).

25 μL samples are taken from the apical solution after ˜2 minutes and atthe end of the experiment. These samples represent donor samples fromthe start and the end of the experiment.

300 μL will be taken from the basolateral (receiver) wells at eachscheduled time point and the post value of TEER is measured at the endthe experiment. To all collected samples acetonitrile will be added to afinal concentration of 50% in the samples. The collected samples will bestored at −20° C. until analysis by HPLC or LC-MS.

Basolateral to Apical Transport

Generally every compound will be tested in 2-4 wells. The basolateraland the apical wells will contain 1.55 mL and 0.4 mL TB, respectively,and the standard concentration of the tested substances is 10 μM.Furthermore all test solutions and buffers will contain 1% DMSO. Priorto the experiment the transport plates are precoated with culture mediumcontaining 10% serum for 30 minutes to avoid nonspecific binding toplastic material.

After 21 to 28 days in culture on filter supports the cells are readyfor permeability experiments. The culture medium from the apical wellsare removed and the inserts are transferred to a wash row (No. 1) in anew plate without inserts (Transport plate).

The transport plate comprises 3 rows of 4 wells. Row 1 is denoted “wash”and row 3 is the “experimental row”. The transport plate has previouslybeen prepared with 1.5 mL TB (pH 7.4) in wash row No. 1 and with 1.55 mLTB (pH 7.4), including the test substance, in experimental row No. 3(donor side).

0.5 mL transport buffer (HBSS, 25 mM MES, pH 6.5) is added to theinserts in row No. 1 and the cell monolayers are equilibrated in thetransport buffer system for 30 minutes, 37° C. in a polymix shaker.After being equilibrated to the buffer system the TEER value is measuredin each well by an EVOM chop stick instrument.

The transport buffer (TB, pH 6.5) is removed from the apical side andthe insert is transferred to row 3 and 400 μL fresh TB, pH 6.5 is addedto the inserts. After 30 minutes 250 μL is withdrawn from the apical(receiver) well and replaced by fresh transport buffer. Thereafter 250μL samples will be withdrawn and replaced by fresh transport bufferevery 30 minutes until the end of the experiment at 120 minutes, andfinally a post value of TEER is measured at the end of the experiment. A25 μL samples will be taken from the basolateral (donor) compartmentafter ˜2 minutes and at the end of the experiment. These samplesrepresent donor samples from the start and the end of the experiment.

To all collected samples acetonitrile will be added to a finalconcentration of 50% in the samples. The collected samples will bestored at −20° C. until analysis by HPLC or LC-MS.

Calculation

Determination of the cumulative fraction absorbed, FA_(cum), versustime. FA_(cum) is calculated from:

${FA}_{cum} = {\sum\frac{C_{RI}}{C_{DI}}}$

Where C_(Ri) is the receiver concentration at the end of the interval iand C_(Di) is the donor concentration at the beginning of interval i. Alinear relationship should be obtained.

The determination of permeability coefficients (P_(app), cm/s) arecalculated from:

$P_{app} = \frac{( {k \cdot V_{R}} )}{( {A \cdot 60} )}$

where k is the transport rate (min⁻¹) defined as the slope obtained bylinear regression of cumulative fraction absorbed (FA_(cum)) as afunction of time (min), V_(R) is the volume in the receiver chamber(mL), and A is the area of the filter (cm²).

Reference Compounds

Category of absorption in man Markers absorption in man (%) PASSIVETRANSPORT Low (0-20%) Mannitol 16 Methotrexate 20 Moderate (21-75%)Acyclovir 30 High (76-100%) Propranolol 90 Caffeine 100 ACTIVE TRANSPORTAmino acid transporter L-Phenylalanine 100 ACTIVE EFFLUX PGP-MDR1Digoxin 30

Greater permeability through the gastrointestinal tissue is advantageousin that it allows for the use of a smaller dose to achieve similarlevels of exposure to a less permeable compound administered in a higherdose. A low dose is advantageous in that it minimizes the cost of goodsfor a daily dose, which is a crucial parameter in a drug which is takenfor protracted time periods.

All references referred to in this application, including patent andpatent applications, are incorporated herein by reference to the fullestextent possible.

Throughout the specification and the claims which follow, unless thecontext requires otherwise, the word ‘comprise’, and variations such as‘comprises’ and ‘comprising’, will be understood to imply the inclusionof a stated integer, step, group of integers or group of steps but notto the exclusion of any other integer, step, group of integers or groupof steps.

The application of which this description and claims forms part may beused as a basis for priority in respect of any subsequent application.The claims of such subsequent application may be directed to any featureor combination of features described herein. They may take the form ofproduct, composition, process, or use claims and may include, by way ofexample and without limitation, the following claims:

1. A compound of the formula I:

0 R⁴N H wherein R^(1a) is H; and R^(1b) is C₁-C₆alkyl, optionallysubstituted with 1-3 substituents independently selected from: halo,hydroxy, cyano, azido, C₁-C₄haloalkyl, C₁-C₄alkoxy, C₁-C₄haloalkoxy,C₁-C₄alkoxycarbonyl, C₁-C₄alkylcarbonyl, amine, C₁-C₄alkylamine,C₁-C₄dialkylamine, C₁-C₄alkylsulfonyl, C₁-C₄alkylsulfonylamino,aminocarbonyl, aminosulphonyl, Carbocyclyl and Het; or R^(1b) isCarbocyclyl or Het; or R^(1a) and R^(1b) together with the N atom towhich they are attached define a saturated cyclic amine with 3-6 ringatoms; wherein the Carbocyclyl, Het or cyclic amine is optionallysubstituted with 1-3 substituents independently selected from halo,hydroxy, cyano, azido, C₁-C₄alkyl, C₁-C₄haloalkyl, C₁-C₄alkoxy,C₁-C₄haloalkoxy, C₁-C₄alkoxycarbonyl, C₁-C₄alkylcarbonyl, amine,C₁-C₄alkylamine, C₁-C₄dialkylamine, C₁-C₄alkylsulfonyl,C₁-C₄alkylsulfonylamino, aminocarbonyl, aminosulphonyl,RxOOC—C₀-C₂alkylene (where Rx is H, C₁-C₄alkyl or C₁-C₄haloalkyl),phenyl, benzyl or C₂-C₆cycloalkylC₀-C₂alkylene; wherein the phenyl,benzyl or cycloalkyl moiety is optionally substituted with 1-3substituents independently selected from halo, C₁-C₄alkyl,C₁-C₄haloalkyl or C₁-C₄alkoxy); R^(2a) and R^(2b) are independentlyselected from H, halo, C₁-C₄alkyl, C₁-C₄haloalkyl, C₁-C₄alkoxy, orR^(2a) and R^(2b) together with the carbon atom to which they areattached form a C₃-C₆cycloalkyl; R³ is a C₅-C₁₀ alkyl, optionallysubstituted with 1-3 substituents independently selected from halo,C₁-C₄haloalkyl, C₁-C₄alkoxy, C₁-C₄haloalkoxy; or R³ is a C₂-C₄alkylchain with at least 2 chloro or 3 fluoro substituents; or R³ isC₃-C₇cycloalkylmethyl, optionally substituted with 1-3 substituentsindependently selected from C₁-C₄alkyl, halo, C₁-C₄haloalkyl,C₁-C₄alkoxy, C₁-C₄haloalkoxy; R⁴ is C₁-C₆alkyl, C₁-C₆haloalkyl,C₁-C₆alkylamino or C₁-C₆dialkylamino; Het is a stable, monocyclic orbicyclic, saturated, partially saturated or aromatic ring systemcontaining 1-4 heteroatoms independently selected from O, S and N, eachring having 5 or 6 ring atoms; Carbocyclyl is C₃-C₆cycloalkyl,C₅-C₆cycloalkenyl or phenyl; or a pharmaceutically acceptable salt,hydrate or N-oxide thereof.
 2. A compound according to claim 1, whereinR^(1b) is methyl, cyclopropyl, 1-phenylethyl, or a 5 or 6 memberedheterocyclic ring containing 1-3 nitrogen atoms and 0 or 1 sulphuratoms, the cyclopropyl, phenyl or heterocyclic ring being optionallysubstituted with up to three substituents independently selected fromC₁-C₄alkyl, halo, C₁-C₄haloalkyl, C₁-C₄alkoxy, C₁-C₄haloalkoxy,C₁-C₄alkoxycarbonyl, C₁-C₄alkylcarbonyl, amine, C₁-C₄alkylamine,C₁-C₄-dialkylamine, C₁-C₄alkylsulfonyl, C₁-C₄alkylsulfonylamino,aminocarbonyl, aminosulphonyl, RxOOC—C₀-C₂alkylene (where Rx is H orC₁-C₄alkyl) or C₃-C₆cycloalkylC₀-C₂alkylene or benzyl (the cycloalkyl,or the phenyl ring of the benzyl being optionally substituted with 1-3substituents selected from C₁-C₄alkyl, halo, C₁-C₄haloalkyl,C₁-C₄alkoxy, C₁-C₄haloalkoxy)
 3. A compound according to claim 2,wherein the heterocyclic ring is pyrrolyl, pyrazolyl, imidazolyl,triazolyl, thiazolyl, or thiadiazolyl, any of which is optionallysubstituted with C₁-C₄alkyl, halo, C₁-C₄haloalkyl, C₁-C₄alkoxy,C₃-C₆cycloalkyl or C₃-C₆cycloalkylmethyl.
 4. A compound according toclaim 3, wherein the heterocyclic ring is pyrrazol-1-yl, optionallysubstituted with C₁-C₄alkyl, halo, C₁-C₄haloalkyl, C₁-C₄alkoxy orcyclopropyl.
 5. A compound according to claim 4, wherein the pyrazolylis N-substituted with C₁-C₄alkyl, such as ethyl or methyl.
 6. A compoundaccording to any of claims 1-5, wherein R^(2a) and R^(2b) are both F. 7.A compound according to any of claims 1-5, wherein one of R^(2a) andR^(2b) is H, and the other is Cl, F, F₃C or MeO.
 8. A compound accordingto any of claims 1-5, wherein R^(2a) and R^(2b) are both H.
 9. Acompound according to any of claims 1-8, wherein R³ is t-butylmethyl,cyclobutylmethyl, 1-methylcyclobutylmethyl or 1-methylcyclopentylmethyl,any of which is optionally substituted with one or two F or MeO.
 10. Acompound according to any of claims 1-8, wherein R³ is1-methylcyclopentylmethyl or 1-fluorocyclopentylmethyl.
 11. A compoundaccording to any of claims 1-10, wherein R⁴ is C₁-C₆alkyl, especiallymethyl or ethyl.
 12. A compound according to any of claims 1-10, whereinR⁴ is C₁-C₆haloalkyl, especially chloroalkyl or fluoroalkyl.
 13. Apharmaceutical composition comprising a compound according to anypreceding claim and a pharmaceutically acceptable vehicle therefor. 14.Use of a compound as defined in any of claims 1-12 for the prophylaxisor treatment of a disorder characterised by inappropriate expression oractivation of cathepsin S.
 15. Use according to claim 14, wherein thedisorder is selected from a) Psoriasis; b) Autoimmune indications,including idiopathic thrombocytopenic purpura (ITP), rheumatoidarthritis (RA), multiple schlerosis (MS), myasthenia gravis (MG),Sjögrens syndrome, Grave's disease and systemic lupus erythematosis(SLE); or c) Non-automimmune indications including allergic rhinitis,asthma, artherosclerosis, chronic obstructive pulmonary disease (COPD)and chronic pain.
 16. A method for the prophylaxis or treatment of adisorder characterised by inappropriate expression or activation ofcathepsin S, the method comprising the administration of an effectiveamount of a compound as defined in any of claims 1-12 to a human oranimal afflicted with or at risk of the disorder.
 17. A method accordingto claim 16, wherein the disorder is a) Psoriasis; b) An autoimmuneindication selected from the group consisting of idiopathicthrombocytopenic purpura (ITP), rheumatoid arthritis (RA), multipleschlerosis (MS), myasthenia gravis (MG), Sjögrens syndrome, Grave'sdisease and systemic lupus erythematosis (SLE); or c) A non-automimmuneindication selected from the group consisting of allergic rhinitis,asthma, artherosclerosis, chronic obstructive pulmonary disease (COPD)and chronic pain.
 18. A compound according to any one of claims 1 to 12,for use as a medicament.
 19. A compound according to claim 18 for use inthe prophylaxis or treatment of a disorder characterised byinappropriate expression or activation of cathepsin S.